Elena Beltramo

Regulation of intracellular glucose and polyol pathway by thiamine and benfotiamine in vascular cells cultured in high glucose

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 μmol/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 chromatography-mass 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.

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.

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 (HUVEC) 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 μmol/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. [Diabetologia (1996) 39: 1263–1268]

Thiamine and benfotiamine prevent increased apoptosis in endothelial cells and pericytes cultured in high glucose

High glucose induces pathological alterations in small and large vessels, possibly through increased formation of AGE, activation of aldose reductase and protein kinase C, and increased flux through the hexosamine pathway. We showed previously that thiamine and benfotiamine correct delayed replication and increase lactate production in endothelial cells subjected to high glucose. We now aim at verifying the effects of thiamine and benfotiamine on cell cycle, apoptosis, and expression of adhesion molecules in endothelial cells and pericytes, under high ambient glucose.

Pericyte Loss in Diabetic Retinopathy: Mechanisms and Consequences

The onset of diabetic retinopathy is characterized by morphologic alterations of the microvessels, with thickening of the basement membrane, loss of inter-endothelial tight junctions and early and selective loss of pericytes, together with increased vascular permeability, capillary occlusions, microaneurysms and, later, loss of endothelial cells (EC). A key role in the evolution of the disease is played by pericytes, specialized contractile mesenchymal cells of mesodermal origin, that, in capillaries, exert a function similar to smooth muscle cells in larger vessels, regulating vascular tone and perfusion pressure. Thickening of the basement membrane, together with systemic and local hypertension, hyperglycaemia, advanced glycation end-product formation and hypoxia, may disrupt the tight link between pericytes and EC causing pericyte apoptosis, while endothelium, deprived of proliferation control, can give rise to new vessels. Pericyte dropout has great consequences on capillary remodelling and may cause the first abnormalities of the diabetic eye which can be observed clinically. Hyperglycaemia and local hypertension are known to be a direct cause of pericyte apoptosis and dropout, and intracellular biochemical pathways of the glucose metabolites have been explored. However, the exact mechanisms are not yet fully understood and need further clarification in order to develop new effective drugs for the prevention of retinopathy.

Effects of thiamine and benfotiamine on intracellular glucose metabolism and relevance in the prevention of diabetic complications

Thiamine (vitamin B1) is an essential cofactor in most organisms and is required at several stages of anabolic and catabolic intermediary metabolism, such as intracellular glucose metabolism, and is also a modulator of neuronal and neuro-muscular transmission. Lack of thiamine or defects in its intracellular transport can cause a number of severe disorders. Thiamine acts as a coenzyme for transketolase (TK) and for the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes, enzymes which play a fundamental role for intracellular glucose metabolism. In particular, TK is able to shift excess fructose-6-phosphate and glycerhaldeyde-3-phosphate from glycolysis into the pentose-phosphate shunt, thus eliminating these potentially damaging metabolites from the cytosol. Diabetes might be considered a thiamine-deficient state, if not in absolute terms at least relative to the increased requirements deriving from accelerated and amplified glucose metabolism in non-insulin dependent tissues that, like the vessel wall, are prone to complications. A thiamine/TK activity deficiency has been described in diabetic patients, the correction of which by thiamine and/or its lipophilic derivative, benfotiamine, has been demonstrated in vitro to counteract the damaging effects of hyperglycaemia on vascular cells. Little is known, however, on the positive effects of thiamine/benfotiamine administration in diabetic patients, apart from the possible amelioration of neuropathic symptoms. Clinical trials on diabetic patients would be necessary to test this vitamin as a potential and inexpensive approach to the prevention and/or treatment of diabetic vascular complications.

Pericyte adhesion is impaired on extracellular matrix produced by endothelial cells in high hexose concentrations.

Abstract.Aims/hypothesis: Thickening of the basement membrane and selective loss of pericytes are early events in diabetic retinopathy. We aimed at checking whether pericyte interaction with extracellular matrix produced by endothelial cells is influenced by the hexose concentrations in which endothelial cells are cultured. Methods: Conditioned extracellular matrixes were obtained by growing human umbilical vein endothelial cells in media containing 28 mmol/l hexoses (d-glucose, d-galactose, l-glucose), which undergo different intracellular processing, before and after adding the inhibitors of protein glycation thiamine or aminoguanidine. Having removed the endothelium, bovine retinal pericytes were grown on such matrixes and, in separate experiments, on laminin, fibronectin or type IV collagen. Pericyte adhesion was determined by cell counts 18 h after seeding. Results: Reduced adhesion was observed on matrixes produced in high d-glucose, high d-galactose and high l-glucose. Both thiamine and aminoguanidine restored impaired pericyte adhesion when added to high d-glucose and high d-galactose, but not l-glucose. Laminin, fibronectin and type IV collagen did not consistently modify pericyte adhesion. Conclusions/interpretations: Pericyte adhesion is impaired on extracellular matrix produced by endothelium in high hexose concentrations. This could result from excess protein glycation, corrected by aminoguanidine and thiamine, rather than altered glycoprotein composition. [Diabetologia (2002) 45: 416–419]

Effects of high glucose and thiamine on the balance between matrix metalloproteinases and their tissue inhibitors in vascular cells

Pericyte survival in diabetic retinopathy depends also on interactions with extracellular matrix (ECM) proteins, which are degraded by matrix metalloproteinases (MMP). Elevated glucose influences ECM turnover, through expression of MMP and their tissue inhibitors, TIMP. We reported on reduced pericyte adhesion to high glucose-conditioned ECM and correction by thiamine. We aimed at verifying the effects of thiamine and benfotiamine on MMP-2, MMP-9 and TIMP expression and activity in human vascular cells with high glucose. In HRP, MMP-2 activity, though not expression, increased with high glucose and decreased with thiamine and benfotiamine; TIMP-1 expression increased with high glucose plus thiamine and benfotiamine; MMP-9 was not expressed. In EC, MMP-9 and MMP-2 expression and activity increased with high glucose, but thiamine and benfotiamine had no effects; TIMP-1 expression was unchanged. Neither glucose nor thiamine modified TIMP-2 and TIMP-3 expression. TIMP-1 concentrations did not change in either HRP or EC. High glucose imbalances MMP/TIMP regulation, leading to increased ECM turnover. Thiamine and benfotiamine correct the increase in MMP-2 activity due to high glucose in HRP, while increasing TIMP-1.

Extracellular vesicles derived from mesenchymal stem cells induce features of diabetic retinopathy in vitro.

AimsLoss of pericytes in the early phases of diabetic retinopathy (DR) may disrupt their stable association with endothelial cells (EC), leading to EC proliferation and, eventually, angiogenesis. Extracellular vesicles (EV) are small membrane particles derived from different cells which contain biologically active proteins and RNA and are known to promote phenotypic changes in target cells. In diabetic-like conditions, EV derived from MSC may play a role in vessel destabilization by interfering with the strict interactions between EC/pericytes and pericyte/extracellular matrix.MethodsWe examined the behaviour of retinal pericytes exposed to EV derived from MSC cultured in physiological and diabetic-like conditions (high glucose and/or hypoxia).ResultsMSC-derived EV are able to enter the pericytes, cause their detachment and migration from the substrate, and increase blood-barrier permeability. Moreover, EV added to EC/pericytes co-cultures in Matrigel promote in vitro angiogenesis. These effects may be mediated by matrix metalloproteinase-2, expressed by both EV and EV-stimulated pericytes, and are exacerbated if MSC are previously cultured in conditions (high glucose and/or hypoxia) mimicking the diabetic microvascular milieu.ConclusionsWe confirm that MSC-derived EV contribute to angiogenesis, showing that they may not only exert a direct stimulus to EC proliferation, but also induce pericyte detachment, thus leaving EC free to proliferate. In addition, we demonstrate a possible link between EV and the early stages of the pathogenesis of DR. Diabetic-like conditions may influence vessel remodelling during angiogenesis through EV paracrine signalling.

Human pericyte–endothelial cell interactions in co-culture models mimicking the diabetic retinal microvascular environment

Pericytes regulate vascular tone, perfusion pressure and endothelial cell (EC) proliferation in capillaries. Thiamine and benfotiamine counteract high glucose-induced damage in vascular cells. We standardized two human retinal pericyte (HRP)/EC co-culture models to mimic the diabetic retinal microvascular environment. We aimed at evaluating the interactions between co-cultured HRP and EC in terms of proliferation/apoptosis and the possible protective role of thiamine and benfotiamine against high glucose-induced damage. EC and HRP were co-cultured in physiological glucose and stable or intermittent high glucose, with or without thiamine/benfotiamine. No-contact model: EC were plated on a porous membrane suspended into the medium and HRP on the bottom of the same well. Cell-to-cell contact model: EC and HRP were plated on the opposite sides of the same membrane. Proliferation (cell counts and DNA synthesis), apoptosis and tubule formation in Matrigel were assessed. In the no-contact model, stable high glucose reduced proliferation of co-cultured EC/HRP and EC alone and increased co-cultured EC/HRP apoptosis. In the contact model, both stable and intermittent high glucose reduced co-cultured EC/HRP proliferation and increased apoptosis. Stable high glucose had no effects on HRP in separate cultures. Both EC and HRP proliferated better when co-cultured. Thiamine and benfotiamine reversed high glucose-induced damage in all cases. HRP are sensitive to soluble factors released by EC when cultured in high glucose conditions, as suggested by conditioned media assays. In the Matrigel models, addition of thiamine and benfotiamine re-established the high glucose-damaged interactions between EC/HRP and stabilized microtubules.

Different apoptotic responses of human and bovine pericytes to fluctuating glucose levels and protective role of thiamine.

Vascular cells in diabetes are subjected to daily fluctuations from high to low glucose. We aimed at investigating whether pulsed exposure to different glucose concentrations influences apoptosis in human retinal pericytes (HRP) versus bovine retinal pericytes (BRP), with consequences on the onset of diabetic retinopathy, and the possible protective role of thiamine.