Elena Berrone

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 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.

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.

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.

Thiamine and benfotiamine prevent apoptosis induced by high glucose-conditioned extracellular matrix in human retinal pericytes.

Early and selective loss of pericytes and thickening of the basement membrane are hallmarks of diabetic retinopathy. We reported reduced adhesion, but no changes in apoptosis, of bovine retinal pericytes cultured on extracellular matrix (ECM) produced by endothelial cells in high glucose (HG). Since human and bovine pericytes may behave differently in conditions mimicking the diabetic milieu, we verified the behaviour of human retinal pericytes cultured on HG‐conditioned ECM.

Detection of cellular prion protein in exosomes derived from ovine plasma

Prion protein (PrP) is present at extremely low levels in the blood of animals and its detection is complicated by the poor sensitivity of current standard methodologies. Interesting results have been obtained with recent advanced technologies that are able to detect minute amounts of the pathological PrP (PrPSc), but their efficiency is reduced by various factors present in blood. In this study, we were able to extract cellular PrP (PrPC) from plasma-derived exosomes by a simple, fast method without the use of differential ultracentrifugation and to visualize it by Western blotting, reducing the presence of most plasma proteins. This result confirms that blood is capable of releasing PrP in association with exosomes and could be useful to better study its role in the pathogenesis of transmissible spongiform encephalopathies.

Retinal heat shock protein 25 in early experimental diabetes

Diabetic retinopathy is the leading cause of blindness in adults, and oxidative stress has been pathogenically associated with retinal neurodegeneration. Cellular stresses induce expression of heat shock proteins (HSPs) and this results in cytoprotection. Our aim was to assess retinal expression of HSP25 in early experimental diabetes. Mice were rendered diabetic by streptozotocin injection. Ten weeks after diabetes onset retinal HSP25 expression were studied by real-time PCR, immunoblotting and immunohistochemistry (IHC). Expression of nitrotyrosine and Cu/Zn superoxide dismutase (SOD), was assessed by IHC and apoptosis by TUNEL. Retinal HSP25 mRNA and protein expression was significantly increased in diabetic as compared to non-diabetic animals and localised predominantly within the retinal ganglion cells (RGC) layer. This was paralleled overexpression of nitrotyrosine and SOD and enhanced apoptosis. In early experimental diabetes, HSP25 is overexpressed in the RGC layer in parallel with markers of oxidative stress and apoptosis.