Glyoxalase 1-knockdown in human aortic endothelial cells - effect on the proteome and endothelial function estimates.

Methylglyoxal (MG), an arginine-directed glycating agent, is implicated in diabetic late complications. MG is detoxified by glyoxalase 1 (GLO1) of the cytosolic glyoxalase system. The aim was to investigate the effects of MG accumulation by GLO1-knockdown under hyperglycaemic conditions in human aortic endothelial cells (HAECs) hypothesizing that the accumulation of MG accounts for the deleterious effects on vascular function. SiRNA-mediated knockdown of GLO1 was performed and MG concentrations were determined. The impact of MG on the cell proteome and targets of MG glycation was analysed, and confirmed by Western blotting. Markers of endothelial function and apoptosis were assessed. Collagen content was assayed in cell culture supernatant. GLO1-knockdown increased MG concentration in cells and culture medium. This was associated with a differential abundance of cytoskeleton stabilisation proteins, intermediate filaments and proteins involved in posttranslational modification of collagen. An increase in fibrillar collagens 1 and 5 was detected. The extracellular concentration of endothelin-1 was increased following GLO1-knockdown, whereas the phosphorylation and amount of eNOS was not influenced by GLO1-knockdown. The expression of ICAM-1, VCAM-1 and of MCP-1 was elevated and apoptosis was increased. MG accumulation by GLO1-knockdown provoked collagen expression, endothelial inflammation and dysfunction and apoptosis which might contribute to vascular damage.

Vascular effects of advanced glycation endproducts: Clinical effects and molecular mechanisms.

The enhanced generation and accumulation of advanced glycation endproducts (AGEs) have been linked to increased risk for macrovascular and microvascular complications associated with diabetes mellitus. AGEs result from the nonenzymatic reaction of reducing sugars with proteins, lipids, and nucleic acids, potentially altering their function by disrupting molecular conformation, promoting cross-linking, altering enzyme activity, reducing their clearance, and impairing receptor recognition. AGEs may also activate specific receptors, like the receptor for AGEs (RAGE), which is present on the surface of all cells relevant to atherosclerotic processes, triggering oxidative stress, inflammation and apoptosis. Understanding the pathogenic mechanisms of AGEs is paramount to develop strategies against diabetic and cardiovascular complications.

Mitochondrial 8-oxoguanine glycosylase decreases mitochondrial fragmentation and improves mitochondrial function in H9C2 cells under oxidative stress conditions.

The mitochondrial DNA base modification 8-hydroxy 2'-deoxyguanine (8-OHdG) is one of the most common DNA lesions induced by reactive oxygen species (ROS) and is considered an index of DNA damage. High levels of mitochondrial 8-OHdG have been correlated with increased mutation, deletion, and loss of mitochondrial (mt) DNA, as well as apoptosis. 8-Oxoguanosine DNA glycosylase-1 (OGG1) recognizes and removes 8-OHdG to prevent further DNA damage. We evaluated the effects of OGG1 on mtDNA damage, mitochondrial function, and apoptotic events induced by oxidative stress using H9C2 cardiac cells treated with menadione and transduced with either Adv-Ogg1 or Adv-Control (empty vector). The levels of mtDNA 8-OHdG and the presence of apurinic/apyrimidinic (AP) sites were decreased by 30% and 35%, respectively, in Adv-Ogg1 transduced cells (P < 0.0001 and P < 0.005, respectively). In addition, the expression of base excision repair (BER) pathway members APE1 and DNA polymerase γ was upregulated by Adv-Ogg1 transduction. Cells overexpressing Ogg1 had increased membrane potential (P < 0.05) and decreased mitochondrial fragmentation (P < 0.005). The mtDNA content was found to be higher in cells with increased OGG1 (P < 0.005). The protein levels of fission and apoptotic factors such as DRP1, FIS1, cytoplasmic cytochrome c, activated caspase-3, and activated caspase-9 were lower in Adv-Ogg1 transduced cells. These observations suggest that Ogg1 overexpression may be an important mechanism to protect cardiac cells against oxidative stress damage.

Impact of GLO1 knock down on GLUT4 trafficking and glucose uptake in L6 myoblasts.

Methylglyoxal (MG), a highly reactive α-dicarbonyl metabolite of glucose degradation pathways, protein and fatty acid metabolism, plays an important role in the pathogenesis of diabetic complications. Hyperglycemia triggers enhanced production of MG and increased generation of advanced glycation endproducts (AGEs). In non-enzymatic reactions, MG reacts with arginine residues of proteins to form the AGEs argpyrimidine and hydroimidazolone. Glyoxalase 1 (GLO1), in combination with glyoxalase 2 and the co-factor glutathione constitute the glyoxalase system, which is responsible for the detoxification of MG. A GLO1 specific knock down results in accumulation of MG in targeted cells. The aim of this study was to investigate the effect of intracellularly accumulated MG on insulin signaling and on the translocation of the glucose transporter 4 (GLUT4). Therefore, L6 cells stably expressing a myc-tagged GLUT4 were examined. For the intracellular accumulation of MG, GLO1, the first enzyme of the glyoxalase pathway, was down regulated by siRNA knock down and cells were cultivated under hyperglycemic conditions (25 mM glucose) for 48 h. Here we show that GLO1 knock down augmented GLUT4 level on the cell surface of L6 myoblasts at least in part through reduction of GLUT4 internalization, resulting in increased glucose uptake. However, intracellular accumulation of MG had no effect on GLUT4 concentration or modification. The antioxidant and MG scavenger NAC prevented the MG-induced GLUT4 translocation. Tiron, which is also a well-known antioxidant, had no impact on MG-induced GLUT4 translocation.

A molecular mechanism for therapeutic effects of cGMP-elevating agents in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive, usually fatal disease with abnormal vascular remodeling. Pulmonary artery smooth muscle cells (PASMCs) from PAH patients are hyperproliferative and apoptosis-resistant and demonstrate decreased signaling in response to bone morphogenetic proteins (BMPs). Cyclic GMP-elevating agents are beneficial in PAH, but their mechanism(s) of action are incompletely understood. Here we show that BMP signaling via Smad1/5/8 requires cGMP-dependent protein kinase isotype I (PKGI) to maintain PASMCs in a differentiated, low proliferative state. BMP cooperation with cGMP/PKGI was crucial for transcription of contractile genes and suppression of pro-proliferative and anti-apoptotic genes. Lungs from mice with low or absent PKGI (Prkg1(+/-) and Prkg1(-/-) mice) exhibited impaired BMP signaling, decreased contractile gene expression, and abnormal vascular remodeling. Conversely, cGMP stimulation of PKGI restored defective BMP signaling in rats with hypoxia-induced PAH, consistent with cGMP-elevating agents reversing vascular remodeling in this PAH model. Our results provide a mechanism for the therapeutic effects of cGMP-elevating agents in PAH and suggest that combining them with BMP mimetics may provide a novel, disease-modifying approach to PAH therapy.

Modulation of dynamin-related protein 1 (DRP1) function by increased O-linked-ß-N-acetylglucosamine modification (O-GlcNAc) in cardiac myocytes.

O-linked-N-acetyl-glucosamine glycosylation (O-GlcNAcylation) of the serine and threonine residues of cellular proteins is a dynamic process and affects phosphorylation. Prolonged O-GlcNAcylation has been linked to diabetes-related complications, including mitochondrial dysfunction. Mitochondria are dynamically remodeling organelles, that constantly fuse (fusion) and divide (fission). An imbalance of this process affects mitochondrial function. In this study, we found that dynamin-related protein 1 (DRP1) is O-GlcNAcylated in cardiomyocytes at threonine 585 and 586. O-GlcNAcylation was significantly enhanced by the chemical inhibition of N-acetyl-glucosaminidase. Increased O-GlcNAcylation decreases the phosphorylation of DRP1 at serine 637, which is known to regulate DRP1 function. In fact, increased O-GlcNAcylation augments the level of the GTP-bound active form of DRP1 and induces translocation of DRP1 from the cytoplasm to mitochondria. Mitochondrial fragmentation and decreased mitochondrial membrane potential also accompany the increased O-GlcNAcylation. In conclusion, this report shows, for the first time, that O-GlcNAcylation modulates DRP1 functionality in cardiac muscle cells.

Regulation of mitochondrial morphology and function by O-GlcNAcylation in neonatal cardiac myocytes.

Mitochondria are crucial organelles in cell life serving as a source of energy production and as regulators of Ca(2+) homeostasis, apoptosis, and development. Mitochondria frequently change their shape by fusion and fission, and recent research on these morphological dynamics of mitochondria has highlighted their role in normal cell physiology and disease. In this study, we investigated the effect of high glucose on mitochondrial dynamics in neonatal cardiac myocytes (NCMs). High-glucose treatment of NCMs significantly decreased the level of optical atrophy 1 (OPA1) (mitochondrial fusion-related protein) protein expression. NCMs exhibit two different kinds of mitochondrial structure: round shape around the nuclear area and elongated tubular structures in the pseudopod area. High-glucose-treated NCMs exhibited augmented mitochondrial fragmentation in the pseudopod area. This effect was significantly decreased by OPA1 overexpression. High-glucose exposure also led to increased O-GlcNAcylation of OPA1 in NCMs. GlcNAcase (GCA) overexpression in high-glucose-treated NCMs decreased OPA1 protein O-GlcNAcylation and significantly increased mitochondrial elongation. In addition to the morphological change caused by high glucose, we observed that high glucose decreased mitochondrial membrane potential and complex IV activity and that OPA1 overexpression increased both levels to the control level. These data suggest that decreased OPA1 protein level and increased O-GlcNAcylation of OPA1 protein by high glucose lead to mitochondrial dysfunction by increasing mitochondrial fragmentation, decreasing mitochondrial membrane potential, and attenuating the activity of mitochondrial complex IV, and that overexpression of OPA1 and GCA in cardiac myocytes may help improve the cardiac dysfunction in diabetes.

Diabetic cardiomyopathy--to take a long story serious.

Being independent of coronary artery disease and hypertension, diabetic cardiomyopathy is a distinct primary disease process, which precedes the development of congestive heart failure. Epidemiologic as well as clinical studies confirmed the close link between diabetes mellitus and heart failure. Altered cardiac structure and function are common diagnoses in patients with type 2 diabetes mellitus. Hyperglycemia leading to the formation of advanced glycation end products and hyperlipidemia resulting in lipotoxicity are of structural and functional impact on cardiac muscle and cardiomyocytes. New and more sensitive methods of diagnosis identify early diastolic dysfunction as a precursor of the development of congestive heart failure. This review focuses on the mechanistic approach to understand the molecular basis of diabetic cardiomyopathy in patients with type 2 diabetes mellitus.

Effects of n-3 fatty acids on macro- and microvascular function in subjects with type 2 diabetes mellitus.

BACKGROUND: Recent evidence supports the protective effects of n-3 (omega-3) fatty acids (n-3 FAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on vascular function. OBJECTIVE: We investigated the effects of EPA and DHA on postprandial vascular function in subjects with type 2 diabetes mellitus. DESIGN: In a double-blind, placebo-controlled, randomized, crossover manner, 34 subjects with type 2 diabetes mellitus received daily either 2 g purified EPA/DHA (termed n-3 FAs) or olive oil (placebo) for 6 wk. At the end of this period, we measured macrovascular (brachial ultrasound of flow-mediated dilatation; FMD) and microvascular [laser-Doppler measurements of reactive hyperemia (RH) of the hand] function at fasting and 2, 4, and 6 h after a high-fat meal (600 kcal, 21 g protein, 41 g carbohydrates, 40 g fat). RESULTS: Fasting vascular function remained unchanged after n-3 FAs and placebo. Postprandial FMD decreased from fasting after placebo, with a maximum decrease (38%) at 4 h-an effect that was significantly reduced (P = 0.03 for time x treatment interaction) by n-3 FA supplementation (maximum decrease in FMD was at 4 h: 13%). RH remained unchanged after placebo, whereas it improved significantly (P = 0.04 for time x treatment interaction) after n-3 FA supplementation (maximum increase was at 2 h: 27%). CONCLUSIONS: In subjects with type 2 diabetes mellitus, 6 wk of supplementation with n-3 FAs reduced the postprandial decrease in macrovascular function relative to placebo. Moreover, n-3 FA supplementation improved postprandial microvascular function. These observations suggest a protective vascular effect of n-3 FAs.

Advanced glycation end products strongly activate platelets.

BACKGROUND: Diabetes mellitus is characterized by hyperglycemia that plays an important role in the pathogenesis of diabetic complications including cardiovascular diseases. Moreover, hyperglycemia induces increased generation of advanced glycation end products (AGEs). The activation of platelets is associated with the development of cardiovascular diseases. AIM OF THE STUDY: The question whether AGEs acutely induce platelet activation as a response to exogenous stimulus is addressed. MATERIALS AND METHODS: The effect of AGEs derived from food and human serum being purified by lysozyme affinity chromatography was examined by incubating in vitro freshly isolated blood platelets from fasted subjects at various concentrations and different time points. Platelet activation, determined as expression of surface markers CD62 and CD63, and the presence of the receptor for AGEs (RAGE) in platelet membranes was measured by flow cytometric analysis using specific antibodies. RESULTS: Incubation with food-derived as well as serum-derived AGEs stimulated significantly the expression of CD62 up to 7.1-fold and CD63 up to 2.2-fold at the platelet surface membrane as a function of concentration and time. Incubation with thrombin or AGEs significantly increased RAGE expression twofold at the platelet surface membrane. CONCLUSIONS: The increase in surface activation marker and RAGE expression in platelets, resulting from concentrations of AGEs that occur in vivo after a meal or a drink as a source of exogenous AGEs, points to signaling mechanisms for food AGEs that could favor the precipitation of acute postprandial ischemic events.

Dietary advanced glycation endproducts and oxidative stress: in vivo effects on endothelial function and adipokines.

Advanced glycation endproducts (AGEs) and oxidative stress (OS) contribute to the development and progression of diabetic complications. We have reported that dietary AGEs and OS induce acute endothelial dysfunction in vivo, but little is known about their effects on adipokines. Twenty inpatients with type 2 diabetes mellitus (mean age: 55.9; range: 32-71 years), received a standard diabetes diet for 6 days. On days 4 and 6, the acute effects of a high-AGE (HAGE) or a low-AGE (LAGE) meal (15.100 vs. 2.750 kU AGE) were studied in a randomized, cross-over, investigator-blinded design. Measurements were performed after an overnight fast, at baseline (B) and at 2, 4, and 6 h after the HAGE or LAGE meals. Both meals had the same ingredients and differed only by the cooking method. Two h following HAGE, a significant decrease from baseline occurred in adiponectin (-10%*double dagger vs. +0%) and leptin (-22%*double dagger vs. -13%*), and a significant increase occurred in vascular cell adhesion molecule 1 (+19%*double dagger vs. -5%) and thiobarbituric acid reactive substances (+23%*double dagger vs. +6%). These changes did not occur, or occurred to a lesser extent, following LAGE. At 4 h following HAGE, an increase in methylglyoxal (+20%double dagger vs. -5%) and E-selectin (+54%*double dagger vs. -3%) occurred. Urinary AGEs increased only after HAGE (+51%*double dagger vs. -2%; values presented as HAGE vs. LAGE; *P < 0.05 vs. baseline, double daggerP < 0.05 vs. LAGE). The postprandial excursions in glucose, insulin, and triglycerides were similar between both meals. A meal rich in AGEs induces acute endothelial and adipocyte dysfunction. These effects were prevented by changing the cooking method.

Effects of low- and high-advanced glycation endproduct meals on macro- and microvascular endothelial function and oxidative stress in patients with type 2 diabetes mellitus.

BACKGROUND: An advanced glycation endproducts (AGEs)-rich diet induces significant increases in inflammatory and endothelial dysfunction markers in type 2 diabetes mellitus (T2DM). OBJECTIVE: The aim was to investigate the acute effects of dietary AGEs on vascular function in T2DM patients. DESIGN: Twenty inpatients with T2DM [x (+/-SEM) age: 55.4 +/- 2.2 y; glycated hemoglobin: 8.8 +/- 0.5%] were investigated. In a randomized crossover design, the effects of a low-AGE (LAGE) and high-AGE (HAGE) meal on macrovascular [by flow-mediated dilatation (FMD)] and microvascular (by Laser-Doppler flowmetry) function, serum markers of endothelial dysfunction (E-selectin, intracellular adhesion molecule 1, and vascular cell adhesion molecule 1), oxidative stress, and serum AGE were assessed. The meals had identical ingredients but different AGE amounts (15.100 compared with 2.750 kU AGE for the HAGE and LAGE meals, respectively), which were obtained by varying the cooking temperature and time. The measurements were performed at baseline and 2, 4, and 6 h after each meal. RESULTS: After the HAGE meal, FMD decreased by 36.2%, from 5.77 +/- 0.65% (baseline) to 3.93 +/- 0.48 (2 h), 3.70 +/- 0.42 (4 h), and 4.42 +/- 0.54% (6 h) (P<0.01 for all compared with baseline). After the LAGE meal, FMD decreased by 20.9%, from 6.04 +/- 0.68% (baseline) to 4.75 +/- 0.48% (2 h), 4.69 +/- 0.51% (4 h), and 5.62 +/- 0.63% (6 h), respectively (P<0.01 for all compared with baseline; P<0.001 for all compared with the HAGE meal). This impairment of macrovascular function after the HAGE meal was paralleled by an impairment of microvascular function (-67.2%) and increased concentrations of serum AGE and markers of endothelial dysfunction and oxidative stress. CONCLUSIONS: In patients with T2DM, a HAGE meal induces a more pronounced acute impairment of vascular function than does an otherwise identical LAGE meal. Therefore, chemical modifications of food by means of cooking play a major role in influencing the extent of postprandial vascular dysfunction.

AGEs and methylglyoxal induce apoptosis and expression of Mac-1 on neutrophils resulting in platelet-neutrophil aggregation.

INTRODUCTION: Diabetes mellitus is characterised by hyperglycaemia that plays an important role in the pathogenesis of diabetic complications including accumulation of methylglyoxal (MG), a highly reactive alpha-dicarbonyl metabolite of glucose degradation pathways and increased generation of advanced glycation end products (AGEs). The aim of this study was to investigate the impact of AGE-BSA, the model substance for AGEs, and MG on cellular haemostasis. MATERIALS AND METHODS: Isolated peripheral blood mononuclear cells (PBMCs) or whole blood was incubated with AGE-BSA and MG. Markers of cellular haemostasis were monitored by flow cytometry. RESULTS: Exposure of PBMCs to AGE-BSA and MG resulted in a dose- and time-dependent increase of TF-expression by monocytes. AGE-BSA and MG induced enhanced platelet-neutrophil aggregation. Examination of platelet activation showed that AGE-BSA induces no direct effect on the expression of P-selectin. However, stimulation with MG resulted in a dose-dependent expression of P-selectin by platelets. Stimulation with AGE-BSA or MG markedly increased dose-dependent expression of Apo2.7 on the neutrophil mitochondria. In addition the analysis demonstrated for the first time that both AGE-BSA and MG induce a dose-dependent expression of the adhesion molecule Mac-1 on the surface of neutrophils. CONCLUSIONS: AGE-BSA as well as MG induced apoptosis of neutrophils and enhanced expression of the adhesion molecule Mac-1 resulting in increased formation of platelet-neutrophil aggregates. These findings may contribute to better understand the mechanism of diabetic thrombosis and the associated high cardiovascular risk of diabetic patients.

Benfotiamine prevents macro- and microvascular endothelial dysfunction and oxidative stress following a meal rich in advanced glycation end products in individuals with type 2 diabetes.

OBJECTIVE: Diabetes is characterized by marked postprandial endothelial dysfunction induced by hyperglycemia, hypertriglyceridemia, advanced glycation end products (AGEs), and dicarbonyls (e.g., methylglyoxal [MG]). In vitro hyperglycemia-induced MG formation and endothelial dysfunction could be blocked by benfotiamine, but in vivo effects of benfotiamine on postprandial endothelial dysfunction and MG synthesis have not been investigated in humans until now. RESEARCH DESIGN AND METHODS: Thirteen people with type 2 diabetes were given a heat-processed test meal with a high AGE content (HAGE; 15.100 AGE kU, 580 kcal, 54 g protein, 17 g lipids, and 48 g carbohydrates) before and after a 3-day therapy with benfotiamine (1,050 mg/day). Macrovascular flow-mediated dilatation (FMD) and microvascular reactive hyperemia, along with serum markers of endothelial disfunction (E-selectin, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1), oxidative stress, AGE, and MG were measured during both test meal days after an overnight fast and then at 2, 4, and 6 h postprandially. RESULTS: The HAGE induced a maximum reactive hyperemia decrease of -60.0% after 2 h and a maximum FMD impairment of -35.1% after 4 h, without affecting endothelium-independent vasodilatation. The effects of HAGE on both FMD and reactive hyperemia were completely prevented by benfotiamine. Serum markers of endothelial dysfunction and oxidative stress, as well as AGE, increased after HAGE. These effects were significantly reduced by benfotiamine. CONCLUSIONS: Our study confirms micro- and macrovascular endothelial dysfunction accompanied by increased oxidative stress following a real-life, heat-processed, AGE-rich meal in individuals with type 2 diabetes and suggests benfotiamine as a potential treatment.