[Advanced glycation end products: A risk factor for human health]. / Les produits de glycation avancée : un risque pour la santé humaine.

Advanced glycation end products (AGE) result from a chemical reaction between the carbonyl group of reducing sugar and the nucleophilic NH2 of a free amino acid or a protein; lysine and arginine being the main reactive amino acids on proteins. Following this first step, a molecular rearrangement occurs, rearrangement of Amadori resulting to the formation of Maillard products. Glycation can cause the clouding of the lens by inducing reactions crosslinking proteins. Specialized receptors (RAGE, Galectin 3…) bind AGE. The binding to the receptor causes the formation of free radicals, which have a deleterious effect because they are powerful oxidizing agents, but also play the role of intracellular messenger, altering the cell functions. This is especially true at the level of endothelial cells: the attachment of AGE to RAGE receptor causes an increase in vascular permeability. AGE binding to endothelium RAGE and to monocytes-macrophages, led to the production of cytokines, growth factors, to the expression of adhesion molecules, and the production of procoagulant activity. Diabetic retinopathy is related to excessive secretion of vascular growth factor (vascular endothelial growth factor [VEGF]). AGE-RAGE receptor binding causes the synthesis and secretion of VEGF. Increased permeability, facilitation of leukocyte migration, the production of reactive oxygen species, cytokines and VEGF suggest that the AGE could be an element of a cascade of reactions responsible for the diabetic angiopathy and vascular damages observed during aging and chronic renal failure. Balanced diet or some drugs can limit the deleterious effect of AGE.

[Molecular basis of red blood cell adhesion to endothelium]. / Bases moléculaires de l'adhérence des globules rouges à l'endothélium.

The extent of red blood cell adhesion is correlated with the incidence of vascular complications and the severity of the disease. Patients with sickle cell anemia (HbSS) experience vasoocclusive episodes. The adhesion of RBCs from HbSS patients is increased and related to VLA-4 exposure, which binds to vascular cell adhesion molecule (VCAM-1). Inter Cellular Adhesion Molecule (ICAM-1), CD31, CD36 and glycans are potential receptors for PfEMP1 of RBCs parasited by plasmodium falciparum. The incidence of vascular complications is very high in patients with diabetes mellitus. RBC adhesion is increased and statistically correlated with the severity of the angiopathy. Glycation of RBC membrane proteins is responsible for binding to the receptor for advanced glycation end products (RAGE). Polycythemia Vera (PV) is the most frequent myeloproliferative disorder and characterized by a high occurrence of thrombosis of mesenteric and cerebral vessels. PV is due to a mutation of the Janus kinase 2 (JAK2 V617F). This mutation stimulates erythropoiesis and is the cause of Lu/BCAM (CD239) phosphorylation, which potentiated the interaction with laminin alpha 5. The couple laminin alpha 5 endothelial and phosphorylated Lu/BCAM explained the increased adhesion of RBCs from patients PV to endothelium.

Endothelial-monocyte activating polypeptide II, a novel antitumor cytokine that suppresses primary and metastatic tumor growth and induces apoptosis in growing endothelial cells.

Neovascularization is essential for growth and spread of primary and metastatic tumors. We have identified a novel cytokine, endothelial-monocyte activating polypeptide (EMAP) II, that potently inhibits tumor growth, and appears to have antiangiogenic activity. Mice implanted with Matrigel showed an intense local angiogenic response, which EMAP II blocked by 76% (P < 0.001). Neovascularization of the mouse cornea was similarly prevented by EMAP II (P < 0.003). Intraperitoneally administered EMAP II suppressed the growth of primary Lewis lung carcinomas, with a reduction in tumor volume of 65% versus controls (P < 0.003). Tumors from human breast carcinoma-derived MDA-MB 468 cells were suppressed by >80% in EMAP II-treated animals (P < 0.005). In a lung metastasis model, EMAP II blocked outgrowth of Lewis lung carcinoma macrometastases; total surface metastases were diminished by 65%, and of the 35% metastases present, approximately 80% were inhibited with maximum diameter <2 mm (P < 0.002 vs. controls). In growing capillary endothelial cultures, EMAP II induced apoptosis in a time- and dose-dependent manner, whereas other cell types were unaffected. These data suggest that EMAP II is a tumor-suppressive mediator with antiangiogenic properties allowing it to target growing endothelium and limit establishment of neovasculature.

Advanced glycation end products regulate extracellular matrix protein and protease expression by human glomerular mesangial cells.

Advanced glycation end products (AGEs) may play a role in the pathogenesis of diabetic nephropathy, by modulating extracellular matrix turnover. AGEs are known to activate specific membrane receptors, including the receptor for AGE (RAGE). In the present study, we analyzed the various receptors for AGEs expressed by human mesangial cells and we studied the effects of glycated albumin and of carboxymethyl lysine on matrix protein and remodelling enzyme synthesis. Membrane RAGE expression was confirmed by FACS analysis. Microarray methods, RT-PCR, and Northern blot analysis were used to detect and confirm specific gene induction. Zymographic analysis and ELISA were used to measure the induction of tPA and PAI-1. We show herein that cultured human mesangial cells express AGE receptor type 1, type 2 and type 3 and RAGE. AGEs (200 microg/ml) induced at least a 2-fold increase in mRNA for 10 genes involved in ECM remodelling, including tPA, PAI-1 and TIMP-3. The increase in tPA synthesis was confirmed by fibrin zymography. The stimulation of PAI-1 synthesis was confirmed by ELISA. AGEs increased PAI-1 mRNA through a signalling pathway involving reactive oxygen species, the MAP kinases ERK-1/ERK-2 and the nuclear transcription factor NF-kappaB, but not AP-1. Carboxymethyl lysine (CML, 5 microM), which is a RAGE ligand, also stimulated PAI-1 synthesis by mesangial cells. In addition, a blocking anti-RAGE antibody partially inhibited the AGE-stimulated gene expression and decreased the PAI-1 accumulation induced by AGEs and by CML. Inhibition of AGE receptors or neutralization of the protease inhibitors TIMP-3 and PAI-1 could represent an important new therapeutic strategy for diabetic nephropathy.

Severity of diabetic microvascular complications is associated with a low soluble RAGE level.

AIMS: The receptor for advanced glycation end-products (RAGE) has been implicated in diabetic microvascular complications, but several lines of evidence suggest that the soluble isoform of RAGE (sRAGE) may protect against AGE-mediated vessel damage. The characterized AGE Nepsilon-carboxymethyllysine (CML) is associated with diabetic microvascular complications. In the present study, we measured blood levels of sRAGE and CML-protein in diabetic patients with and without microvascular complications. METHODS: Thirty patients with type-2 diabetes were recruited into the study, comprising 20 who had no microvascular complications, and 10 who had both retinal and renal complications. sRAGE was measured in serum by ELISA, and CML by competitive ELISA. RESULTS: sRAGE blood levels were similar in both the controls and diabetic patients without microvascular complications. In patients with complications, the mean sRAGE blood level was significantly decreased (1068+/-231pg/mL) compared with diabetic patients without complications (P=0.028). CML-protein was increased in all diabetic patients, but to a higher extent in those who had microvascular complications. CONCLUSION: The association of low sRAGE with high CML-protein levels in diabetic patients who developed severe diabetic complications supports the hypothesis that sRAGE protects vessels against AGE-mediated diabetic microvascular damage.

Red cell and endothelial Lu/BCAM beyond sickle cell disease.

Recent studies shed new lights on the biological function of blood group antigens, such as the adhesion properties of the Lutheran (Lu) blood group antigens carried by the Lu/BCAM glycoproteins. The Lu/BCAM adhesion glycoproteins were first identified as laminin-10/11 erythroid receptors involved in RBC adhesion to endothelium in sickle cell anemia. Lu/BCAM mediated cell adhesion to laminin is stimulated by epinephrine, a physiological stress mediator, and is dependent of phosphorylation by protein kinase A. More recently, we demonstrated that constitutive phosphorylation of Lu/BCAM is also involved in abnormal RBC adhesion to endothelium in patients with polycythemia vera (PV), a frequent myeloproliferative disorders associated with the V617F mutation of the tyrosine kinase JAK2 leading to continuous stimulation of erythropoiesis. This observation suggests that Lu/BCAM could participate to the high incidence of vascular thrombosis that also characterizes PV disease. In mice, which do not express Lu/BCAM in erytroid tissues, invalidation of the Lu/BCAM gene provided evidence that Lu/BCAM gps, as laminin-alpha5 receptors, are involved in vivo in the maintenance of normal basement membrane organization in different non erythroid tissues since up to 90% of the mutant kidney glomeruli exhibited a reduced number of visible capillary lumens and irregular thickening of the glomerular basement membrane, while intestine exhibited smooth muscle coat thickening and disorganization. All these results further illustrate that minor blood group antigens might have important role under physiological and physiopathological conditions in erythroid and non erythroid tissues as well.

Role of Lu/BCAM in abnormal adhesion of sickle red blood cells to vascular endothelium.

Lutheran (Lu) blood group and Basal Cell Adhesion Molecule (BCAM) antigens are both carried by two glycoprotein (gp) isoforms of the immunoglobulin superfamily representing receptors for laminin alpha5 chain. They are expressed in red blood cells, in endothelial cells of vascular capillaries and in epithelial cells of several tissues. Lu/BCAM gps are overexpressed in sickle red blood cells (SS RBCs). Stimulation of SS RBCs by epinephrine activates the PKA depending signaling pathway and induces reinforced Lu/BCAM-mediated adhesion to laminin10/11. We have analyzed the phosphorylation state of Lu/BCAM long isoform cytoplasmic tail and showed that it is phosphorylated by CKII, GSK3b and PKA. Phosphorylation of this isoform in transfected K562 cells is stimulated by effectors of the PKA pathway and induces cell adhesion to laminin10/11. Lu/BCAM gps are highly expressed in endothelial cells and exhibit potential integrin binding motifs. We showed that they interact with integrin alpha4beta1, the unique integrin expressed on the surface of young reticulocytes. Adhesion assays under flow conditions showed that SS RBCs adhere to primary human endothelial cells (HUVEC) after selective activation of intergin alpha4beta1 and that this adhesion is mediated by endothelial Lu/BCAM gps. Our studies show that Lu/BCAM gps expressed either on erythroid or on endothelial cells are involved in SS RBC-endothelium interactions and could play a role in the abnormal adhesion of SS RBCs to vascular endothelium contributing to the vaso-occlusive crises reported for sickle cell disease patients.

Receptor-mediated endothelial cell dysfunction in diabetic vasculopathy. Soluble receptor for advanced glycation end products blocks hyperpermeability in diabetic rats.

Dysfunctional endothelium is associated with and, likely, predates clinical complications of diabetes mellitus, by promoting increased vascular permeability and thrombogenicity. Irreversible advanced glycation end products (AGEs), resulting from nonenzymatic glycation and oxidation of proteins or lipids, are found in plasma, vessel wall, and tissues and have been linked to the development of diabetic complications. The principal means through which AGEs exert their cellular effects is via specific cellular receptors, one of which, receptor for AGE (RAGE), is expressed by endothelium. We report that blockade of RAGE inhibits AGE-induced impairment of endothelial barrier function, and reverse, in large part, the early vascular hyperpermeability observed in diabetic rats. Inhibition of AGE- and diabetes-mediated hyperpermeability by antioxidants, both in vitro and in vivo, suggested the central role of AGE-RAGE-induced oxidant stress in the development of hyperpermeability. Taken together, these data support the concept that ligation of AGEs by endothelial RAGE induces cellular dysfunction, at least in part by an oxidant-sensitive mechanism, contributing to vascular hyperpermeability in diabetes, and that RAGE is central to this pathologic process.

[Aging: role and control of glycation]. / Vieillissement: rôle et contrôle de la glycation.

PURPOSE: Advanced glycation end-products (AGEs) accumulate in aging tissues and organs during rheumatoid arthritis and Alzheimer disease. These aging toxins are especially involved in cell alteration during diabetes mellitus (glycotoxin) and renal failure (uremic toxin). AGEs participate to the endothelial dysfunction leading to diabetic macro but also micro-angiopathy. AGEs binding to cell receptors are critical steps in the deleterious consequences of AGE excess. AGE-receptor activation altered cell and organ functions by a pro-inflammatory, pro-coagulant and pro-fibrosis factors cell response. CURRENT KNOWLEDGE AND KEY POINTS: Non-enzymatic glycation and glycoxidation with glucose auto-oxidation represent the two main pathways resulting in AGE formation. No exclusive AGE classification is actually available. Pathophysiological mechanisms are described to explain AGE toxicity. AGEs bind to cell receptors inducing deleterious consequences such as endothelial dysfunction after endothelial RAGE activation. AGEs can also have deleterious effects through glycated protein accumulation or in situ protein glycation. FUTURE PROSPECTS AND PROJECTS: Many in vitro or animal studies demonstrated that AGE deleterious effects can be prevented by glycation inhibitors, AGE cross-link breakers or AGE-RAGE interaction inhibition. New molecules are actually studied as new strategy to prevent or treat the deleterious effects of these aging toxins.

Postprandial hyperglycemia alters inflammatory and hemostatic parameters.

Glucose or glucose derived products are increased in blood during the postprandial phase and are, to a certain extent, related to meal composition. Glucose and glucose derived products such as advanced glycation end products (AGEs) can be formed in the intracellular compartment but can also be absorbed as AGEs or AGE precursors present in food. Glucose, glucose metabolites and AGEs alter endothelial cell functions, induce adhesion molecule overexpression (ICAM-1, VCAM), cytokine release (IL-6, MCP-1) and tissue factor production. Tumor necrosis factor alpha systemic level is increased during the postprandial phase as are augmented C reactive protein and fibrinogen level. Hyperglycemia induced an increase in plasminogen activator inhibitor, and shortened fibrinogen half life. Hyperglycemia and AGEs provoked an oxidant stress. The formation of reactive oxygen intermediates perturbates NO (Nitric oxide) formation and are deleterious for cell functions. All the modifications observed in the postprandial phase are not too deleterious but their iterative characteristics may lead to vascular dysfunction.

Role of membrane sialic acid content in the adhesiveness of aged erythrocytes to human cultured endothelial cells.

Following our previous observation that the oldest normal red blood cells were the most adherent to human cultured endothelial cells, we attempted to simulate this age-related adherence. Among all the membrane modifications experienced by erythrocytes during their life-span, loss of sialic acids has attracted considerable attention. Using two different preparations of neuraminidase, we performed a sialic acid depletion on the youngest erythrocytes to reach a sialic acid content similar to that observed in physiologically aged erythrocytes. These pretreated youngest cells displayed limited increase in the adhesiveness to endothelial cells, lower than that found with intact oldest cells. To obtain an adhesiveness of pretreated cells similar to that of naturally aged cells, it was necessary to exceed 80% of sialic acid depletion. At this extent of desialation, modifications of the electrophoretic pattern of glycophorins were observed as well as the appearance of peanut agglutinin reactivity which were never found in physiologically aged erythrocytes. Therefore, the sialic acid loss cannot be considered as being a single determinant factor of the naturally aged red cell adhesiveness.

Liposomes bearing platelet proteins: a model for surface functions studies.

An improved procedure for the direct transfer of membrane proteins from human platelets to liposomes involving the treatment of platelets with linolenic acid was developed. The transfer of platelet proteins to liposomes prepared from the mixture of L-alpha-dimyristoyl-phosphatidylcholine/sphingomyelin in the molar ratio 80/20 appeared to be significantly enhanced compared with liposomes prepared from the same components mixed in other ratios. A wide range of platelet proteins was transferred, the most important being GPIb (170 kDa), GPIIb/IIIa (135 and 110 kDa). GPIV (90 kDa), GPIX (24 kDa) and the serotonin transporter (68 kDa). The recognition interactions between these proteoliposomes and specific protein antibodies clearly indicate that the non-invasive procedure used in this study ensured the reproducible transfer of platelet proteins without essentially altering their original conformation. The obtained results reveal also that the affinity of proteoliposomes to bind paroxetin was virtually the same as that of the native serotonin transporter. These results provide an indication of the possible use of such proteoliposomes as models to study at the molecular level the interaction of these proteins with their ligands.

Acute modulation of albumin microvascular leakage by advanced glycation end products in microcirculation of diabetic rats in vivo.

Advanced glycation end products (AGEs) are nonenzymatic glycosylated adducts of proteins that accumulate in vascular tissue during diabetes and aging. The aim of this work was to study the role of AGEs and of the oxidative mechanisms in diabetes-induced changes in vascular permeability. Intravital videomicroscopy was used to study albumin microvascular leakage in cremaster muscle. The extravasation of a fluorescent macromolecular tracer (fluorescein isothiocyanate-albumin) was measured for 1 h and, after computer-aided image analysis, was expressed as variations of normalized gray levels (arbitrary units). Extravasation of the macromolecular tracer was much higher in diabetic rats than in control rats (slope of extravasation versus time increased by >100%, P < 10(-4)). This increase was significantly inhibited when we blocked AGEs binding to their endothelial receptor by intravenous bolus of soluble recombinant receptor to AGEs (rR-RAGE) (slope of extravasation versus time decreased by 19, 30, and 40%, for 0.5, 2.5, and 5.15 mg/kg rR-RAGE, respectively) or by a 6 mg/kg intravenous bolus of antibody against RAGE (slope decreased by 53%). Systemic injection of probucol (an antioxidant) also significantly inhibited the increase in the extravasation of the macromolecular tracer occurring in experimental diabetes (slope decreased by 51%, P < 10(-4)). These results strongly suggest that in experimental diabetes the interaction of circulating AGEs and endothelial RAGE mediates albumin micro-vascular leakage, possibly via AGE-RAGE-dependent enhanced oxidant stress.

RAGE: a novel cellular receptor for advanced glycation end products.

Exposure of proteins to reducing sugars results in nonenzymatic glycation with the ultimate formation of advanced glycation end products (AGEs). One means through which AGEs modulate cellular functions is through binding to specific cell surface acceptor molecules. The receptor for AGEs (RAGE) is such a receptor and is a newly identified member of the immunoglobulin superfamily expressed on endothelial cells (ECs), mononuclear phagocytes (MPs), and vascular smooth muscle cells (SMCs) in both vivo and in vitro. Binding of AGEs to RAGE results in induction of cellular oxidant stress, as exemplified by the generation of thiobarbituric acid-reactive substances, expression of heme oxygenase type I, and activation of the transcription factor NF-kB, with consequences for a range of cellular functions. AGEs on the surface of diabetic red cells enhance binding to endothelial RAGE and result in enhanced oxidant stress in the vessel wall. By using reagents to selectively block access to RAGE, the role of this receptor in AGE-mediated perturbation of cellular properties can be dissected in detail.

[Indications for transfusions of labile blood products]. / Indications des transfusions de produits sanguins labiles.

Indications for transfusions of red blood cells (RBC) are anemias, which can occur after trauma, in surgery, in obstetrics or oncohematology wards. The main criteria to administer RBC transfusion are hemoglobin level and clinical features. Transfusions are rare when the hemoglobin level is above 10 g/dL and are frequent when it is below 6 g/dL. However clinical setting, patient age, associated diseases, cardiovascular complications are taken into account. Immunocompatibility should always be tested and the transfusion consequences checked immediately and on the long term. Platelet transfusions are performed when the platelet count is low and patients suffered from hemorrhage. In oncohematology patients, platelet transfusion are administered with prophylaxis when the platelet count is lower than 10 g/L. Fresh frozen plasma has now a limited use, only in complex haemostatic disorders and in hemolytic uremic syndrome.

[Electronic learning: interactive learning in medicine or Socrates in electronic guise]. / Electronic learning: apprentissage interactif en médecine ou Socrate en habits électroniques.

E-learning has been widely used for training in different fields. More recently, it was introduced during medical studies or for continuous medical education. The Canadian Universities are pioneers in e-learning creating special departments dedicated to pedagogy. Developing countries like Brazil or Central Europe have made some pilot experiments, which were successful. Several electronic companies have given a free access to the programmes and sites. The use of electronic media leads to an adaptation of teaching methods making them more interactive.

[French training program for medical students in transfusion medicine. Transfusion Medicine Teachers' College]. / Programme en transfusion des étudiants en médecine. Collège des enseignants en transfusion sanguine.

In France, transfusion medicine training program has been updated. A national committee of professors in transfusion medicine propose a series of 13 items which represent the minimum knowledge that general practitioners should possess. This overview of transfusion medicine is far below the level that specialists should reach and they will need an additional specialized training. Several French universities have set up their own training program which is quite similar to the work of the committee of professors. The following recommendations are not strict guidelines but is a common basis which will be improved in 2005 according to new evidence based transfusion medicine.

Colchicine disposition in human leukocytes after single and multiple oral administration.

Inasmuch as leukocytes were reported to be an active pharmacologic compartment, colchicine disposition was determined in plasma, granulocytes, and mononuclear cells in healthy volunteers after 1 mg oral single and multiple doses. After the single dose, maximal colchicine concentration was observed at 1 hour in plasma and 47 hours later in leukocytes. This delay was confirmed by the slow accumulation of colchicine by lymphocytes in culture. In the multiple-dose study, mean granulocyte colchicine concentration (20 to 53 ng/10(9) cells) were twofold higher than in mononuclear cells (9 to 24 ng/10(9) cells). Mean predicted colchicine multiple-dose granulocyte and mononuclear cell concentrations were 2.5-fold and ninefold higher, respectively, than those measured. After the last dose, colchicine decreased, with half-life values between 41 and 46 hours for leukocytes and 49 hours for plasma. This study validates leukocytes as a microcompartment whose kinetics correlates with colchicine biologic effects.

Tumour necrosis factor-alpha, interleukin-1 beta and interleukin-6 in patients with renal cell carcinoma.

Patients with renal cell carcinoma (RCC) can exhibit fever, weight loss and increases in acute phase proteins. Interleukin (IL)-1, tumour necrosis factor (TNF) and IL-6 are considered major mediators of local and systemic inflammation. We measured plasma IL-1 beta, TNF-alpha (immunoradiometric assay) and IL-6 (ELISA) in 78 consecutive patients with untreated RCC and in 56 normal subjects. IL-6 plasma levels were higher in patients with RCC (mean 24.2 pg/ml, 11.1-37.3, 95% confidence interval) than in normal subjects (11.6 pg/ml, 10.1-13.1, n = 39, P < 0.01). The patients with fever or weight loss had higher blood levels of IL-6. IL-6 blood levels were also higher in patients with lymph node invasion and/or distant metastases (94.7 pg/ml, 39.0-150.4, n = 15) than in patients with undisseminated RCC (7.4, 4.1-10.7, n = 63, P < 0.0001). An abnormal IL-6 plasma value (> 40 pg/ml) had a positive predictive value of 91.0% for lymph node and/or metastatic spread of RCC. IL-6 was statistically correlated with C-reactive protein (nephelometric assay) blood values r' = 0.67, n = 78, P < 0.001). The TNF-alpha and IL-1 beta levels were not significantly different in patients with or without fever or weight loss. The plasma levels of the three cytokines were not correlated with the size of the primary tumour. An increased plasma value of IL-6 is a good marker for tumour dissemination in patients with untreated RCC.

IgG binding to endothelial cells in systemic lupus erythematosus.

Endothelial-associated IgG were determined in 20 patients with systemic lupus erythematosis (SLE)-8 of whom had a lupus anticoagulant (LA) and 6 a history of thrombosis. The binding of IgG present in patient plasma to cultured human endothelial cells was detected using radiolabeled staphylococcal protein A. Thirteen samples gave positive results and a significant association between endothelial-associated IgG and lupus anticoagulant was found (p less than 0.05). No statistically significant relationship with a previous history of thrombosis was found. These results suggest that the lupus anticoagulant may be directly involved in immune vascular injury induced by either antibodies or immune complexes in SLE.