Erythrocytes: Central Actors in Multiple Scenes of Atherosclerosis.

The development and progression of atherosclerosis (ATH) involves lipid accumulation, oxidative stress and both vascular and blood cell dysfunction. Erythrocytes, the main circulating cells in the body, exert determinant roles in the gas transport between tissues. Erythrocytes have long been considered as simple bystanders in cardiovascular diseases, including ATH. This review highlights recent knowledge concerning the role of erythrocytes being more than just passive gas carriers, as potent contributors to atherosclerotic plaque progression. Erythrocyte physiology and ATH pathology is first described. Then, a specific chapter delineates the numerous links between erythrocytes and atherogenesis. In particular, we discuss the impact of extravasated erythrocytes in plaque iron homeostasis with potential pathological consequences. Hyperglycaemia is recognised as a significant aggravating contributor to the development of ATH. Then, a special focus is made on glycoxidative modifications of erythrocytes and their role in ATH. This chapter includes recent data proposing glycoxidised erythrocytes as putative contributors to enhanced atherothrombosis in diabetic patients.

Enhanced oxidative stress and damage in glycated erythrocytes.

Diabetes is associated with a dramatic mortality rate due to its vascular complications. Chronic hyperglycemia in diabetes leads to enhanced glycation of erythrocytes and oxidative stress. Even though erythrocytes play a determining role in vascular complications, very little is known about how erythrocyte structure and functionality can be affected by glycation. Our objective was to decipher the impact of glycation on erythrocyte structure, oxidative stress parameters and capacity to interact with cultured human endothelial cells. In vitro glycated erythrocytes were prepared following incubation in the presence of different concentrations of glucose. To get insight into the in vivo relevance of our results, we compared these data to those obtained using red blood cells purified from diabetics or non-diabetics. We measured erythrocyte deformability, susceptibility to hemolysis, reactive oxygen species production and oxidative damage accumulation. Altered structures, redox status and oxidative modifications were increased in glycated erythrocytes. These modifications were associated with reduced antioxidant defence mediated by enzymatic activity. Enhanced erythrocyte phagocytosis by endothelial cells was observed when cultured with glycated erythrocytes, which was associated with increased levels of phosphatidylserine-likely as a result of an eryptosis phenomenon triggered by the hyperglycemic treatment. Most types of oxidative damage identified in in vitro glycated erythrocytes were also observed in red blood cells isolated from diabetics. These results bring new insights into the impact of glycation on erythrocyte structure, oxidative damage and their capacity to interact with endothelial cells, with a possible relevance to diabetes.

Aging and glycation promote erythrocyte phagocytosis by human endothelial cells: Potential impact in atherothrombosis under diabetic conditions.

BACKGROUND AND AIMS: Atherothrombotic plaques of type 2 diabetic (T2D) patients are characterized by an increased neovascularization and intraplaque hemorrhage. The clearance of erythrocytes may be carried out by vascular cells. We explored the potential of human endothelial cells to bind and phagocyte in vitro aged and/or glycated erythrocytes as well as erythrocytes obtained from diabetic patients. METHODS: Fresh, aged and glycated-aged erythrocytes from healthy volunteers and T2D patients were tested for their binding and phagocytosis capacity as well as the potential functional consequences on endothelial cells (viability, proliferation and wound healing capacity). Immunohistochemistry was also performed in human carotid atherothrombotic samples (from patients with or without T2D). RESULTS: Aging and glycation of erythrocytes induced phosphatidylserine (PS) exposure and oxidative stress leading to enhanced endothelial cell binding and engulfment. Phagocytosis by endothelial cells was more pronounced with aged and glycated erythrocytes than with fresh ones. Phagocytosis was enhanced with T2D versus healthy erythrocytes. Furthermore, endothelial wound healing potential was significantly blunted after exposure to glycated-aged versus fresh erythrocytes. Finally, we show that interactions between erythrocytes and endothelial cells and their potential phagocytosis may occur in vivo, in atherothrombotic conditions, in neovessels and in the luminal endothelial lining. CONCLUSIONS: Endothelial cells may play an important role in erythrocyte clearance in an atherothrombotic environment. Under diabetic conditions, erythrocyte glycation favors their engulfment by endothelial cells and may participate in endothelial dysfunction, thereby promoting vulnerable atherothrombotic plaques to rupture.

Enhanced effects of curcumin encapsulated in polycaprolactone-grafted oligocarrageenan nanomicelles, a novel nanoparticle drug delivery system.

One of the most effective strategies to enhance the bioavailability and the therapeutic effect of hydrophobic drugs is the use of nanocarriers. We have used κ-carrageenan extracted from Kappaphycus alvarezii to produce oligocarrageenan via an enzymatic degradation process. Polycaprolactone (PCL) chains were grafted onto the oligocarrageenans using a protection/deprotection technique yielding polycaprolactone-grafted oligocarrageenan. The resulting amphiphilic copolymers formed spherical nanomicelles with a mean size of 187 ± 21 nm. Hydrophobic drugs such as curcumin were efficiently encapsulated in the micelles and released within 24-72 h in solution. The micelles were non-cytotoxic and facilitated the uptake of curcumin by endothelial EA-hy926 cells. They also increased the anti-inflammatory effect of curcumin in TNF-alpha-induced inflammation experiments. Finally, in vivo experiments supported a lack of toxicity in zebrafish and thus the potential use of polycaprolactone-grafted oligocarrageenan to improve the delivery of hydrophobic compounds to different organs, including liver, lung and brain as shown in mice.

Circulating Concentrations of Redox Biomarkers Do Not Improve the Prediction of Adverse Cardiovascular Events in Patients With Type 2 Diabetes Mellitus.

BACKGROUND: Despite pathophysiological relevance and promising experimental data, the usefulness of biomarkers of oxidative stress for cardiac risk prediction is unclear. The aim of our study was to investigate the prognostic value of 6 biomarkers exploring different pathways of oxidative stress for predicting adverse cardiovascular outcomes in patients with type 2 diabetes mellitus beyond established risk factors. METHODS AND RESULTS: The SURDIAGENE (Survie, Diabete de type 2 et Genetique) prospective cohort study consecutively recruited 1468 patients with type 2 diabetes mellitus. Assays were performed at baseline, and incident cases of major adverse cardiovascular events (MACE)-first occurrence of cardiovascular death, nonfatal myocardial infarction, or stroke-were recorded during a median of 64 months. Advanced oxidation protein products, oxidative hemolysis inhibition assay, ischemia-modified albumin, and total reductive capacity of plasma were not associated with the risk of MACE in univariate analyses. Fluorescent advanced glycation end products and carbonyls were associated with MACE (hazard ratio=1.38 per SD, 95% confidence interval 1.24-1.54, P<0.001 and hazard ratio=1.15 per SD, 95% confidence interval 1.04-1.27, P=0.006, respectively) in univariate analysis, but when added to a multivariate predictive model including traditional risk factors for MACE, these markers did not significantly improve c-statistics or integrated discrimination index of the model. CONCLUSIONS: These plasma concentrations of 6 markers, which cover a broad spectrum of oxidative processes, were not significantly associated with MACE occurrence and were not able to improve MACE risk discrimination and classification beyond classical risk factors in type 2 diabetes mellitus patients.

Glycation of human serum albumin impairs binding to the glucagon-like peptide-1 analogue liraglutide.

The long-acting glucagon-like peptide-1 analogue liraglutide has proven efficiency in the management of type 2 diabetes and also has beneficial effects on cardiovascular diseases. Liraglutide's protracted action highly depends on its capacity to bind to albumin via its palmitic acid part. However, in diabetes, albumin can undergo glycation, resulting in impaired drug binding. Our objective in this study was to assess the impact of human serum albumin (HSA) glycation on liraglutide affinity. Using fluorine labeling of the drug and 19F NMR, we determined HSA affinity for liraglutide in two glycated albumin models. We either glycated HSA in vitro by incubation with glucose (G25- or G100-HSA) or methylglyoxal (MGO-HSA) or purified in vivo glycated HSA from the plasma of diabetic patients with poor glycemic control. Nonglycated commercial HSA (G0-HSA) and HSA purified from plasma of healthy individuals served as controls. We found that glycation decreases affinity for liraglutide by 7-fold for G100-HSA and by 5-fold for MGO-HSA compared with G0-HSA. A similarly reduced affinity was observed for HSA purified from diabetic individuals compared with HSA from healthy individuals. Our results reveal that glycation significantly impairs HSA affinity to liraglutide and confirm that glycation contributes to liraglutide's variable therapeutic efficiency, depending on diabetes stage. Because diabetes is a progressive disease, the effect of glycated albumin on liraglutide affinity found here is important to consider when diabetes is managed with this drug.

A hemorrhagic transformation model of mechanical stroke therapy with acute hyperglycemia in mice.

Clinical benefit for mechanical thrombectomy (MT) in stroke was recently demonstrated in multiple large prospective studies. Acute hyperglycemia (HG) is an important risk factor of poor outcome in stroke patients, including those that underwent MT. The aim of this therapy is to achieve a complete reperfusion in a short time, given that reperfusion damage is dependent on the duration of ischemia. Here, we investigated the effects of acute HG in a mouse model of ischemic stroke induced by middle cerebral artery occlusion (MCAO). Hyperglycemic (intraperitoneal [ip] injection of glucose) and control (ip saline injection) 10-week male C57BL6 mice were subjected to MCAO (30, 90, and 180 min) followed by reperfusion obtained by withdrawal of the monofilament. Infarct volume, hemorrhagic transformation (HT), neutrophil infiltration, and neurological scores were assessed at 24 hr by performing vital staining, ELISA immunofluorescence, and behavioral test, respectively. Glucose injection led to transient HG (blood glucose = 250-390 mg/dL) that significantly increased infarct volume, HT, and worsened neurological outcome. In addition, we report that HG promoted blood-brain barrier disruption as shown by hemoglobin accumulation in the brain parenchyma and tended to increase neutrophil extravasation within the infarcted area. Acute HG increased neurovascular damage for all MCAO durations tested. HTs were observed as early as 90 min after ischemia under hyperglycemic conditions. This model mimics MT ischemia/reperfusion and allows the exploration of brain injury in hyperglycemic conditions.

Diabetes-induced hepatic oxidative stress: a new pathogenic role for glycated albumin.

Increased oxidative stress and advanced glycation end-product (AGE) formation are major contributors to the development of type 2 diabetes. Here plasma proteins e.g. albumin can undergo glycoxidation and play a key role in diabetes onset and related pathologies. However, despite recent progress linking albumin-AGE to increased oxidative stress and downstream effects, its action in metabolic organs such as the liver remains to be elucidated. The current study therefore investigated links between oxidative perturbations and biochemical/structural modifications of plasma albumin, and subsequent downstream effects in transgenic db/db mouse livers and HepG2 cells, respectively. Our data reveal increased oxidative stress biomarkers and lipid accumulation in plasma and livers of diabetic mice, together with albumin glycoxidation. Purified mouse albumin modifications resembled those typically found in diabetic patients, i.e. degree of glycation, carbonylation, AGE levels and in terms of chemical composition. Receptor for AGE expression and reactive oxygen species production were upregulated in db/db mouse livers, together with impaired proteolytic, antioxidant and mitochondrial respiratory activities. In parallel, acute exposure of HepG2 cells to glycated albumin also elicited intracellular free radical formation. Together this study demonstrates that AGE-modified albumin can trigger damaging effects on the liver, i.e. by increasing oxidative stress, attenuating antioxidant capacity, and by impairment of hepatic proteolytic and respiratory chain enzyme activities.

Curcuma longa polyphenols improve insulin-mediated lipid accumulation and attenuate proinflammatory response of 3T3-L1 adipose cells during oxidative stress through regulation of key adipokines and antioxidant enzymes.

Plant polyphenols may exert beneficial action against obesity-related oxidative stress and inflammation which promote insulin resistance. This study evaluated the effect of polyphenols extracted from French Curcuma longa on 3T3-L1 adipose cells exposed to H2 O2 -mediated oxidative stress. We found that Curcuma longa extract exhibited high amounts of curcuminoids identified as curcumin, demethoxycurcumin, and bisdemethoxycurcumin, which exerted free radical-scavenging activities. Curcuma longa polyphenols improved insulin-mediated lipid accumulation and upregulated peroxisome proliferator-activated receptor-gamma gene expression and adiponectin secretion which decreased in H2 O2 -treated cells. Curcuminoids attenuated H2 O2 -enhanced production of pro-inflammatory molecules such as interleukin-6, tumor necrosis factor-alpha, monocyte chemoattractant protein-1, and nuclear factor κappa B. Moreover, they reduced intracellular levels of reactive oxygen species elevated by H2 O2 and modulated the expression of genes encoding superoxide dismutase and catalase antioxidant enzymes. Collectively, these findings highlight that Curcuma longa polyphenols protect adipose cells against oxidative stress and may improve obesity-related metabolic disorders. © 2016 BioFactors, 42(4):418-430, 2016.

Oxidative damage in diabetics: insights from a graduate study in La Reunion University.

Due to the growing incidence of diabetes in developed nations, there is a compelling case to be made for teaching graduate students more deeply about mechanisms underlying this disease. Diabetes is associated with enhanced oxidative stress and protein glycation via the covalent binding of glucose molecules. Albumin represents the major plasmatic protein and undergoes enhanced glycoxidative modifications in diabetic condition. La Réunion Island, a French department located in the Indian Ocean exhibit a growing incidence of diabetes. At the University of La Réunion, our research group named GEICO (Groupe d'Etude sur l'Inflammation Chronique et l'Obésité) participated to foster research and training in diabetes context and focuses on the impact of glycated albumin mediated oxidative stress on cell physiopathology. A laboratory course was designed by our group to introduce graduate students to cutting edge techniques in redox biology while providing insights into scientific processes and methods. This two weeks research laboratory training took place at CYROI, a local biotechnology center that provides advanced facilities for research, business, and education. Using histochemistry, molecular biology, biochemical techniques, student investigated oxidative damages in liver from leptin receptor deficient diabetic mice compared to control littermates. In addition, they used an in vitro model by assaying oxidative impact of glycated albumin on hepatoma carcinoma HepG2 cells. This article gives an overview of the organization and protocol used by the students during their two weeks training in the laboratory. Therefore, it may be helpful for teaching graduate students techniques used in research laboratory working on redox biology.

Evaluation of maximal O2 uptake with undergraduate students at the University of La Reunion.

The maximal rate of O2 consumption (VO2 max) constitutes one of the oldest fitness indexes established for the measure of cardiorespiratory fitness and aerobic performance. Procedures have been developed in which VO2 max is estimated from physiological responses during submaximal exercise. Generally, VO2 max is estimated using the classical renowned Astrand-Ryhming test. In young adults, poor fitness and low aerobic performance are often associated with a sedentary lifestyle, which is a well-described factor for the development of obesity and its related disorders such as cardiovascular diseases and type 2 diabetes. In the Indian Ocean, the inhabitants of La Reunion Island, a French overseas department, exhibit an increasing prevalence of obesity and type 2 diabetes. At the University of La Reunion, a new laboratory course involving students was designed to teach the indirect evaluation of their VO2 max from the classical Astrand-Ryhming test and using a cycle ergometer as the exercise mode. Inverse and significant correlations were established between the students' fat mass percentages and their VO2 max and between their waist-to-hip ratio and VO2 max as well. Results from the international physical activity questionnaire showed that most participants in this laboratory were sedentary students. Therefore, this laboratory makes the students practice and understand the use of a classical test to estimate their VO2 max. It also alerts them to the correlation between a sedentary lifestyle and higher body fat content. This exercise allowed students to use a scientific method to engage the problem of sedentary lifestyle, which is a real world issue.