Acrylamide induces accelerated endothelial aging in a human cell model.

Acrylamide (AAM) has been recently discovered in food as a Maillard reaction product. AAM and glycidamide (GA), its metabolite, have been described as probably carcinogenic to humans. It is widely established that senescence and carcinogenicity are closely related. In vitro, endothelial aging is characterized by replicative senescence in which primary cells in culture lose their ability to divide. Our objective was to assess the effects of AAM and GA on human endothelial cell senescence. Human umbilical vein endothelial cells (HUVECs) cultured in vitro were used as model. HUVECs were cultured over 3 months with AAM or GA (1, 10 or 100 µM) until growth arrest. To analyze senescence, ß-galactosidase activity and telomere length of HUVECs were measured by cytometry and semi-quantitative PCR, respectively. At all tested concentrations, AAM or GA reduced cell population doubling compared to the control condition (p < 0.001). ß-galactosidase activity in endothelial cells was increased when exposed to AAM (≥10 µM) or GA (≥1 µM) (p < 0.05). AAM (≥10 µM) or GA (100 µM) accelerated telomere shortening in HUVECs (p < 0.05). In conclusion, in vitro chronic exposure to AAM or GA at low concentrations induces accelerated senescence. This result suggests that an exposure to AAM might contribute to endothelial aging.

M1 and M2 macrophage proteolytic and angiogenic profile analysis in atherosclerotic patients reveals a distinctive profile in type 2 diabetes.

This study aimed to investigate atherosclerotic mediators' expression levels in M1 and M2 macrophages and to focus on the influence of diabetes on M1/M2 profiles. Macrophages from 36 atherosclerotic patients (19 diabetics and 17 non-diabetics) were cultured with interleukin-1ß (IL-1ß) or IL-4 to induce M1 or M2 phenotype, respectively. The atherosclerotic mediators' expression was evaluated by quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results showed that M1 and M2 macrophages differentially expressed mediators involved in proteolysis and angiogenesis processes. The proteolytic balance (matrix metalloproteinase-9 (MMP-9)/tissue inhibitor of metalloproteinase-1 (TIMP-1), MMP-9/plasminogen activator inhibitor-1 (PAI-1) and MMP-9/tissue factor pathway inhibitor-2 (TFPI-2) ratios) was higher in M1 versus M2, whereas M2 macrophages presented higher angiogenesis properties (increased vascular endothelial growth factor/TFPI-2 and tissue factor/TFPI-2 ratios). Moreover, M1 macrophages from diabetics displayed more important proangiogenic and proteolytic activities than non-diabetics. This study reveals that M1 and M2 macrophages could differentially modulate major atherosclerosis-related pathological processes. Moreover, M1 macrophages from diabetics display a deleterious phenotype that could explain the higher plaque vulnerability observed in these subjects.

Alterations in neutrophil production and function at an early stage in the high-fructose rat model of metabolic syndrome.

BACKGROUND: Although neutrophils are crucially involved in inflammation, they have received only little attention in metabolic syndrome (MetS). We hypothesized that neutrophil infiltration into adipose tissue (AT) may occur at an early stage of MetS, in association with modulation of major functions of neutrophils and of their bone marrow production. METHODS: Fifty-six male Sprague-Dawley rats were fed regular (control rats (CRs)) or high-fructose (60%; fructose-fed rats (FFRs)) diets. After 6 weeks, metabolic parameters were measured. Distribution of neutrophils into AT was investigated by immunohistochemistry. Function of circulating neutrophils (activation, reactive oxygen species production, phagocytosis, and apoptosis) was determined by flow cytometry. Granulopoiesis was evaluated by measuring the number and survival characteristics of neutrophil progenitors using bone marrow culture assays and flow cytometry. RESULTS: Compared with the CR group, the FFR group developed MetS (i.e., arterial hypertension, hypertriglyceridemia, fasting hyperglycemia, and greater intra-abdominal AT volume) and presented higher neutrophil infiltration into AT. At resting state, no significant difference for circulating neutrophil functions was observed between the 2 groups. In contrast, circulating neutrophils from the FFR group exhibited higher responses to phorbol-12-myristate-13-acetate for all studied functions, compared with the CR group, suggesting that early MetS induces neutrophil priming. In parallel, a diminished clonal capacity and an increased apoptosis in bone marrow-derived granulocyte progenitors and neutrophil precursors were observed in the FFR group compared with the CR group. CONCLUSIONS: These results provide evidence of an increased infiltration into intra-abdominal AT and modified production, function, and phenotype of neutrophils at an early stage of high-fructose diet-induced MetS.

Soluble form of receptor for advanced glycation end-products (sRAGE): do sRAGE ligands or anti-sRAGE auto-antibodies interfere with sRAGE quantification?

BACKGROUND: The soluble form of the receptor for advanced glycation end-products (sRAGE) has been studied in various diseases. It is not clear why sRAGE levels vary between studies, with controversial results. What also remains to be determined is whether receptor for advanced glycation end-products (RAGE) ligands could affect sRAGE assessment by epitope masking. Recently described anti-sRAGE autoantibodies may play an interfering role. The aim of this study was therefore to investigate the influence of RAGE ligands and anti-sRAGE autoantibodies on sRAGE quantification. METHODS: The RAGE ligands carboxymethyllysine (CML; AGEs with a high affinity for RAGE), S100 proteins, high-mobility group protein B1 (HMGB1) and ß-amyloid peptide (aß) were tested by enzyme-linked immunosorbent assay (ELISA) with recombinant sRAGE (rHu-sRAGE) or serum from healthy controls. Using ELISA, anti-sRAGE autoantibodies (IgGs) were identified in haemodialysis (HD) patients, then purified and incubated with rHu-sRAGE or serum to investigate their effects on sRAGE levels. RESULTS: RAGE ligands, either alone at three different concentrations (CML was also tested at different glycation levels) or a mixture of all these ligands, did not affect sRAGE levels when incubated with rHu-sRAGE or control serum. Compared with healthy controls, HD patients had higher levels of sRAGE (P < 0.001) and anti-sRAGE IgGs (P < 0.05). However, incubation of rHu-sRAGE with purified IgGs from HD patients had no effect on sRAGE quantification. CONCLUSIONS: RAGE ligands or anti-sRAGE autoantibodies did not interfere with sRAGE quantification. Further studies are required to elucidate the variability in sRAGE levels reported in the literature and to define the potential of sRAGE for use as a reliable biomarker.

Plasma glycation levels are associated with severity in sepsis.

BACKGROUND: Advanced glycation end-products (AGE) have been involved in inflammatory diseases and may have an important role in the progression of symptoms. However, few studies have analysed the levels of glycated proteins in sepsis. In this study, we evaluated the levels of the well-known AGE (N(ε) -(carboxymethyl)lysine (CML) and N(ε) -(carboxyethyl)lysine (CEL)) in the plasma of septic patients. MATERIAL AND METHODS: Plasma from 36 patients admitted to an adult intensive care unit and 6 healthy controls had the levels of CML/CEL measured by ELISA. RESULTS: The level of AGE in plasma decreased with the increase of severity (1·40±0·46 nmol/mg of protein in sepsis, 0·58±0·23 nmol/mg of protein in severe sepsis and 0·31±0·12 nmol/mg of protein in septic shock). Control plasma presented low AGE concentration (0·06±0·01 nmol/mg protein). Also, we found a decrease in plasma AGE in those patients that died at the end of 28 days follow-up (0·80±0·50 nmol/mg of protein in survivors vs. 0·31±0·10 nmol/mg of protein in nonsurvivors), being associated with the renal component of sequential organ failure assessment (SOFA) score. In the same line, there was a decrease in plasma AGE with the increase in SOFA. CONCLUSIONS: Our data demonstrate that plasma AGE levels are inversely associated with the severity of sepsis and may be associated with kidney dysfunction.

Increased 3-nitrotyrosine levels in mitochondrial membranes and impaired respiratory chain activity in brain regions of adult female rats submitted to daily vitamin A supplementation for 2 months.

Vitamin A supplementation among women is a common habit worldwide in an attempt to slow aging progression due to the antioxidant potential attributed to retinoids. Nonetheless, vitamin A elicits a myriad of side effects that result from either therapeutic or inadvertent intake at varying doses for different periods. The mechanism behind such effects remains to be elucidated. In this regard, we performed the present work aiming to investigate the effects of vitamin A supplementation at 100, 200, or 500IU/kgday(-1) for 2 months on female rat brain, analyzing tissue lipid peroxidation levels, antioxidant enzyme activities (both Cu/Zn-superoxide dismutase - SOD - and Mn-SOD); glutathione S-transferase (GST) and monoamine oxidase (MAO) enzyme activity; mitochondrial respiratory chain activity and redox parameters in mitochondrial membranes, as well as quantifying α- and ß-synucleins, ß-amyloid peptide(1-40), immunoglobulin heavy-chain binding protein/78kDa glucose-regulated protein (BiP/GRP78), receptor for advanced glycation end products (RAGE), D2 receptor, and tumor necrosis factor-α (TNF-α) contents in rat frontal cortex, hippocampus, striatum, and cerebellum. We observed increased lipid peroxidation marker levels, altered Cu/Zn-SOD and Mn-SOD enzyme activities, mitochondrial nitrosative stress, and impaired respiratory chain activity in such brain regions. On the other hand, we did not find any change in MAO and GST enzyme activities, and on α- and ß-synucleins, ß-amyloid peptide(1-40), GRP78/BiP, RAGE, D2 receptor, and TNF-α contents. Importantly, we did not observed any evidence regarding an antioxidant effect of such vitamin at low doses in this experimental model. The use of vitamin A as an antioxidant therapy among women needs to be reexamined.

The effects of vitamin A supplementation to rats during gestation and lactation upon redox parameters: increased oxidative stress and redox modulation in mothers and their offspring.

Vitamin A is an essential nutrient required in adequate amounts for reproduction and development. Subtle variations in the status of maternal nutrition may affect physiological and metabolic parameters in the fetus. Evidence suggests a key role for oxidative stress in these events. Literature is controversial about the effects of vitamin A supplementation. Here, we studied the effects of vitamin A supplementation on female Wistar rats during gestation and lactation on oxidative stress parameters of maternal and offspring tissues. Rats received daily doses of vitamin A at 2500, 12,500 and 25,000IU/kg. We observed an increase of oxidative damage markers in the reproductive tissues and plasma of dams. The activity of glutathione-S-transferase was modulated by vitamin A supplementation. It was found to be increased in the liver of dams and decreased in the kidneys of mothers and offspring. In pups, supplementation decreased the total antioxidant potential of the liver along with decreased superoxide dismutase/catalase activity ratio in the kidney. The levels of lipoperoxidation were increased in male offspring, but decreased in female pups. Collectively, the results suggest that excessive vitamin A intake during gestation and lactation might be toxic for mothers with adverse effects for the developing offspring.

Oxidative damage in the liver of rats treated with glycolaldehyde.

Liver diseases are often associated with hyperglycemia, inflammation, and oxidative stress. These conditions, commonly associated with diabetes mellitus and obesity, facilitate the formation of advanced glycation end products (AGEs). These products are known to impair protein function and promote inflammation. Accumulation of AGEs such as N(ε)-(carboxymethyl)lysine (CML) is related to chronic liver diseases and their severity. Although several reports suggest a crucial role of AGEs in liver failure, there is little investigation on the direct effects of reducing sugars, precursors of AGEs, and on the onset and progression of liver failure. In this work, we investigate the effects of intravenously administrated glycolaldehyde (GA), a short-chain aldehyde, on oxidative parameters in the liver of Wistar rats. Animals received a single injection of GA (10, 50, or 100 mg/kg) and were sacrificed after 6, 12, or 24 hours. Levels of protein carbonyl, lipid peroxidation, and reduced thiol were quantified. The activities of catalase, superoxide dismutase, and glyoxalase I were also assessed. The amount of CML was quantified with specific antibody. There was an increase in oxidative stress markers in the liver of GA-treated rats. Glycolaldehyde induced a decrease in the activities of all enzymes assayed. Also, all tested doses led to an increase in CML content. Our data suggest that GA might play an important role in liver diseases through the impairment of antioxidant defenses and generation of AGEs.

Glycolaldehyde induces oxidative stress in the heart: a clue to diabetic cardiomyopathy?

Cardiovascular complications account for 80% of the mortality related to diabetes mellitus. Hyperglycemia is believed to be the major culprit of angiopathy and cardiomyopathy. High glucose levels and oxidative stress cause elevation of Advanced Glycation End-products that are known to contribute to diabetic complications and correlate with many diseases. However, there are few reports describing the effects of glycating agents other than glucose. Here, we aimed to evaluate the effects of glycolaldehyde (GA) on oxidative stress parameters in the heart of Wistar rats. Male Wistar rats received a single injection of GA (10, 50 or 100 mg/Kg) and were sacrificed 6, 12 or 24 h after injection. As indexes of oxidative stress, we quantified protein carbonylation, lipid peroxidation and total reduced thiols. The activities of superoxide dismutase, catalase and glyoxalase I were assayed. Also, the content of N (ɛ)-(carboxymethyl)lysine (CML) was quantified. Glycolaldehyde induced an imbalance in the redox status, with increased protein carbonylation and lipoperoxidation. Catalase and glyoxalase I had a decrease in their activities. Despite the oxidative stress, we observed no increase in CML content. These results suggest that short-chain aldehydes such as GA might have a significant role in the development of diabetic cardiomyopathy.

Circulating glycolaldehyde induces oxidative damage in the kidney of rats.

Renal failure is a key pathological issue in diabetic patients. Increased levels of advanced glycation end-products (AGEs) have been associated to diabetic complications, including diabetic nephropathy. Models of AGE-treated animals have been applied to evaluate the effect of such molecules on oxidative parameters involved in the pathogenesis and evolution of diabetes disease. However, little is known about the effect of glycating agents other than glucose. Here we investigate the effect of intravenously administrated glycolaldehyde (GA) on oxidative stress parameters of the kidney. Male Wistar rats received a single injection of GA in different doses (10, 50 or 100mg/kg) and were sacrificed after 6, 12 or 24h. Activities of antioxidant enzymes catalase, superoxide dismutase and glyoxalase I were assayed. Damage to proteins and lipids were also assayed. The content of N(epsilon)-(carboxymethyl)lysine (CML) was quantified. Glycolaldehyde induced a decrease in the activity of all enzymes studied. Lipoperoxidation and protein carbonylation raised, accompanied by a decrease in sulfhydryl groups. Despite the oxidative stress generated by GA, no change was found in the content of CML, suggesting that accumulation of AGEs in the kidney might occur at later steps in the development of diabetic nephropathy.

Short-term vitamin A supplementation at therapeutic doses induces a pro-oxidative state in the hepatic environment and facilitates calcium-ion-induced oxidative stress in rat liver mitochondria independently from permeability transition pore formation : detrimental effects of vitamin A supplementation on rat liver redox and bioenergetic states homeostasis.

There is a growing body of evidence showing that vitamin A induces toxic effects in several experimental models and in human beings. In the present work, we have investigated the effects of short-term vitamin A supplementation on the adult rat liver redox status. We have found that vitamin A at therapeutic doses induces a hepatic oxidative insult. Furthermore, we have observed increased antioxidant enzyme activity in the liver of vitamin-A-treated rats. Additionally, some mitochondrial dysfunction was found since superoxide anion production was increased in vitamin-A-treated rat liver submitochondrial particles, which may be the result of impaired mitochondrial electron transfer chain activity, as assessed here. We have also isolated rat liver mitochondria and challenged it with 75 muM CaCl2, a non-oxidant agent that is able to induce mitochondrial oxidative stress indirectly. We have found that mitochondria isolated from vitamin-A-treated rat liver are more sensitive to CaCl2 than control mitochondria regarding the redox status. Importantly, vitamin A seems to alter mitochondrial redox status independently of the participation of the mitochondrial permeability transition pore, which is activated by Ca2+ ions since cyclosporin A did not prevent the oxidative insult elicited by Ca2+ addition. Overall, we show here that mitochondria are a target of vitamin-A-associated toxicity also in vivo.

Antioxidant activities and free radical scavenging potential of Bauhinia microstachya (RADDI) MACBR. (Caesalpinaceae) extracts linked to their polyphenol content.

Aqueous and hydro-ethanolic extracts of Bauhinia microstachya leaves (AEBM and HEBM) were investigated for their phenolic content and phytochemical profile (by spectrophotometry and HPLC), and for their antioxidant activities and free radical scavenging potential in different in vitro systems (TRAP, TEAC, TBARS, nitric oxide, superoxide and hydroxyl radical). HEBM presented a 27.4% higher content of phenolics when compared to AEBM and a distinct phytochemical profile was observed. Our work suggests that both extracts have potent antioxidant activities and that their antioxidant capacity and efficiency vary according to the radical-generating system. In general, HEBM was more effective than AEBM in avoiding ROS-generating damage and in scavenging the various radicals formed. Nevertheless, when results were normalized to total phenolic content, a different profile of antioxidant activities and free radical scavenging potential was observed, particularly against oxidative lipid damage and superoxide radical. B. microstachya extracts may be considered an interesting source of natural antioxidants as well as other phenolic-rich plants.