Erythrocyte adhesion to the vascular endothelium]

Blood cells are in continuous contact with the vascular endothelium. Endothelial cell culture, intravital videomicroscopy allowed the investigation of blood cell-endothelium interactions in dynamic conditions. In the various diseases, diabetes mellitus, sickle cell anemia and malaria, erythrocytes have an increased adhesion to endothelial cells. The presence of advanced glycation end products (AGE) on erythrocytes of diabetics is responsible for their binding to the receptor RAGE present on the endothelium. The AGE-RAGE binding provokes an oxidant stress and induces the expression of the adhesion molecule. Furthermore, erythrocyte AGE induce an increase in vascular permeability. In sickle cell anemia, the increased adhesiveness and the sickling of red blood cells are responsible for thrombosis. Plasmodium falciparum infestation of erythrocytes induces knob formation at the cell surface and the P. falciparum protein binding to CD36, ICAM-1 and thrombospondin present on the endothelium, and facilitates the parasite dissemination.     

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.     

Adhesion of diabetic or sickle cell erythrocyte populations to human endothelium in culture

Erythrocyte adhesion to endothelium was measured using human endothelial cells in culture and a radiometric technique. Erythrocyte adhesion was found to be significantly increased in diabetes mellitus and sickle cell anemia. In both diseases the extent of adhesion was correlated with the clinical severity of the disease. Using 3H Leucine radio-labelled reticulocytes or red cells separated by density gradient according to their age it was possible to further investigate the red cell abnormality responsible for increased adhesion. A population of abnormal reticulocytes in sickle cell anemia exhibited a higher adhesion than the whole red cell population. Diabetic dense red cells (old red cells) appeared to be mostly responsible for the increase in erythrocyte adhesion to endothelium observed in diabetes mellitus.     

The α4β1 integrin in sickle cell disease

The α4β1 integrin is an adhesion receptor expressed on reticulocytes in sickle cell disease (SCD) and mediates the adhesion of these cells to sub-endothelial matrix proteins and the endothelium. In this review, we describe the mechanism of activation of the α4β1 integrin on sickle reticulocytes and discuss novel roles for this integrin in SCD as a result of this activation. We also illustrate novel therapies in SCD that may target the integrin and alleviate vaso-occlusion.     

Adhesion of sickle red cells to endothelium: Myths and future directions

Sickle RBC are abnormally adherent to vascular endothelial cells. We briefly review the mechanisms that underlie this type of cell/cell adhesion, expose a number of extant myths about RBC adhesion, and discuss some aspects that need consideration via future experimentation. The relationship of this phenomenon of RBC-endothelial adhesion to the cytoadherence of parasitized sickle RBC is not yet clear.     

Conception, élaboration et caractérisation de matériaux bioactifs

One promising strategy to control the interactions between biomaterial surfaces and attaching cells involves grafting of adhesion peptides as RGD peptides (R: arginine; G: glycine; D: aspartic acid) to materials on which protein adsorption, which mediates unspecific cell adhesion, is essentially suppressed. This review gives an overview of RGD modified materials, that have been used for cell adhesion, and provides information about technical aspects of RGD immobilization on materials. The impacts of RGD peptide surface density, spatial arrangement as well as integrin affinity and selectivity on cell responses like adhesion and migration are discussed. We have tried to relate one of numerous scientifics adventures initiated by Charles Baquey within our laboratory. This review is dedicated to him for his enthusiasm in the development of project and for his wish of always leading of a professional blooming of his students.     

Conduite in vitro d'une étude de biocompatibilité pour la confection d'un substitut vasculaire bioartificiel de petit calibre

Le travail présenté rapporte une démarche, à savoir les différentes étapes d'une évaluation dans la perspective de l'élaboration d'un substitut vasculaire artificiel hybride, concept qui prévoit d'associer dans une même unité fonctionnelle une composante artificielle d'origine synthétique ou naturelle et une composante cellulaire: l'endothélium vasculaire. Les résultats des essais cliniques les plus récents rapportés dans la littérature, à condition que le revêtement endothélial du substitut soit complet au moment de l'implantation, permettent d'envisager l'utilisation de telles structures cellularisées. Les différentes étapes de cette étude comportent: (1) la sélection d'un matériau propice à l'endothélialisation in vitro par des études de cytocompatibilité, à l'aide de cellules endothéliales en culture; (2) l'étude du phénotype de la composante biologique endothéliale au contact des matériaux sélectionnés (mesures de la production et de la fonctionnalité de deux protéines-clé de la régulation de la balance hémostatique, quantification d'une molécule d'adhésion exprimée à la membrane de la cellule endothéliale, intervenant dans le recrutement des leucocytes en cas de processus inflammatoire; (3) l'étude du devenir du néo-endothélium obtenu, sous contraintes de cisaillement, par quantification de la radioactivité après radiomarquage in situ de la monocouche cellulaire.     

Vascular biosynthesis of nitric oxide: effect on hemostasis and fibrinolysis

Nitric oxide (NO) is a multifunctional effector molecule that plays a central role in the regulation of vascular homeostasis. NO is synthesized from L-arginine by a family of enzymes called NO synthases. The principal source of NO in the vascular system of healthy mammals is the constitutively expressed NO synthase in endothelial cells. The basal endothelial formation of NO can be increased by receptor-dependent agonists (i.e., bradykinin) in a calcium-calmodulin-dependent manner, and also by physical forces (i.e., shear stress), predominantly without changes in the intracellular concentration of free calcium. Nitric oxide can diffuse toward the blood vessel wall where the major target is the smooth muscle cell. NO regulates vascular tone, and the free radical is also a potent inhibitor of smooth muscle cell proliferation, migration and synthesis of extracellular matrix proteins. NO can also diffuse toward the lumen of the blood vessel where it helps maintain blood fluidity. NO inhibits platelets' and leucocytes' adhesion to endothelial cells. In addition, NO inhibits platelet aggregation and facilitates the dissolution of small platelet aggregates. However, the regulatory action of NO on blood cells is most likely limited to the luminal surface of endothelial cells since NO is rapidly scavenged by hemoglobin in erythrocytes and inactivated by oxygen-derived radicals such as superoxide anions. NO can also affect the fibrinolytic activity by regulating the release of tissue-type plasminogen activator and plasminogen activator inhibitor-1. The crucial role of vascular NO in the control of blood fluidity has been demonstrated by the regulation of the bleeding time in humans.     

Role and regulation of sickle red cell interactions with other cells: ICAM-4 and other adhesion receptors

Erythrocytes containing primarily hemoglobin S (SS RBCs) are abnormally adherent. We now know that SS RBCs express numerous adhesion molecules, and that many of these can undergo activation. SS RBCs exposed briefly to epinephrine show markedly increased adhesion to both laminin and endothelial cells. In vivo, infusion of epinephrine-activated but not unstimulated SS RBCs causes RBC adhesion, vaso-occlusion, organ trapping, and shortened RBC survival in the circulation. Epinephrine treatment of SS RBCs before infusion also induces adhesion of murine leukocytes to vascular walls. Indeed, in vitro, SS RBCs can activate leukocyte adhesion and cytokine production. We now have demonstrated both in vitro and in vivo evidence for the importance of RBC signaling and have also shown that SS RBC adhesion is determined by genetic polymorphisms in the signaling pathway that activates adhesion. These advances will hopefully lead to new therapeutic modalities for sickle cell disease.     

Protection against severe clinical manifestations of Plasmodium falciparum malaria among sickle cell trait subjects is due to modification of the release of cytokines and/or cytoadherence of infected erythrocytes to the host vascular beds

It is proposed that the surface ligands of Plasmodium falciparum infected HbAS erythrocytes, not like infected HbAA erythrocytes, are altered due to the sickling that soon takes place once a HbAS erythrocyte gets infected with P. falciparum parasite. This alteration modulates cytoadherence and/or binding of the sickled erythrocytes to the peripheral blood mononuclear cells (PBMCs). Both cytoadherence and binding to PBMCs are responsible for the pathogenesis of malaria. Therefore, subjects of the HbAS genotype experience mild symptoms of malaria. The hypothesis could be tested in vitro by comparing the binding of P. falciparum infected HbAS and HbAA erythrocytes to platelet-endothelial cell adhesion molecule-1 (CD31) and by comparing the levels of tumor necrosis factor (TNF) and interferon gamma (IFN-gamma) following in vitro stimulation of PBMCs by HbAS and HbAA infected erythrocytes.     

Mouse models of sickle cell disease

In the absence of a natural animal model for sickle cell disease, transgenic mouse models have been generated to better understand the complex pathophysiology of the disease and to evaluate potential specific therapies. In the early nineties, the simple addition of human globin genes induced the expression of hemoglobin S (HbS) or HbS-related human hemoglobins in mice still expressing mouse hemoglobin. To increase the proportion of human hemoglobin and the severity of the mouse sickle cell syndrome, the proportion of mouse hemoglobin could be decreased by a combination of mouse alpha- and beta-thalassemic defects, leading to complex genotypes and mild disease. Following the discovery of gene targeting in the mouse embryonic stem cells (ES cells), it was made possible to knock out all mouse adult globin genes (2alpha and 2beta) and to add the human homologous genes elsewhere in the mouse genome. In addition, the human gamma gene of fetal hemoglobin was protecting the fetus from HbS polymer formation. Accordingly, the resulting adult mouse models obtained in 1997, expressing human HbS-only, had a very severe anemia (Hb=5-6 g/dL). In order to survive, these "HbS-only mice" had to reduce the HbS concentration within the red blood cells. The phenotype could be less severe by adding modified human gamma genes, still expressed in adult mice. In 2006, a last "S-only" model was obtained by homologous knock in, replacing the mouse globin genes by human genes. This array of models contributes to better understand the role of different interacting factors in the complexity of sickle cell events, such as red cell defects, changes in blood flow and vaso-occlusion, hyperhemolysis, vascular tone dysregulation, oxidations, inflammation, activation and adhesion of cells, ischemia, reperfusion... In addition, each model has an appropriate usefulness to evaluate experimental therapies in vivo and to perform preclinical studies.     

Pathophysiology of acute thoracic syndrome

Pulmonary complications are the leading cause of morbidity and mortality in sickle cell disease patients. Acute chest syndrome (ACS), in which chest pain and dyspnea, occurs in combination with a recent chest radiograph abnormality, raises both diagnostic and therapeutic challenges. The pathogenesis of ACS involves alterations in blood rheology, increased coagulability, and, above all, increased adhesion of sickle cells to the vascular endothelium and nitric oxide-mediated dysregulation of vascular reactivity. Sickle cell disease thus impacts all the cells in the vascular environment. Recently gained insights into pathophysiology offer hope that new treatments for preventing and treating acute and chronic pulmonary complications will soon become available.     

Anti-CD9 antibodies augment neutrophil adherence to endothelium.

Anti-CD9 antibodies which bind to the CD9 (p24) antigen are known to induce platelet and pre-B-cell aggregation. We show here that human endothelium expresses the CD9 antigen, and anti-CD9 antibodies incubated with endothelium induce a rapid increase in adhesion of neutrophils to endothelium by an action on the endothelial cell. This augmented adhesion is not mediated by glycoprotein IIb/IIIa or by the leucocyte integrins. Binding of anti-CD9 antibody to CD9 induces shedding of the CD9/anti-CD9 complex off the endothelial cell in a time-dependent manner. It is likely that CD9 binding to its ligand induces an activation event within the endothelial cell, resulting in surface expression of a pre-formed adhesive ligand for the neutrophil, or an activation change to a constitutively expressed ligand to render it functional.     

Sickle red cell adhesion: Many issues and some answers

Among multiple pathologies associated with sickle cell disease, sickle red cell-endothelial interaction has been implicated as a potential initiating mechanism in vaso-occlusive events that characterize this disease. Vast literature exists on various aspects of sickle red cell adhesion, but many issues remain unresolved, especially pertaining to the role of sickle red cell heterogeneity, the relative role of multiple adhesion mechanisms and targets of antiadhesive therapy. This review briefly analyzes these issues.     

Erythroid adhesion molecules in sickle cell disease: Effect of hydroxyurea

In sickle cell disease, the complex scenario of vaso-occlusive crisis (VOC) typical of this disease is clearly multifactorial and not fully understood. Cell-cell and cell-cell matrix interactions mediated by adhesive molecules present on blood cells and endothelial cells (ECs) are thought to play an important role. Early studies have shown that sickle red blood cells (RBCs) are abnormally adherent to ECs and some of the molecules involved in these interactions have been identified, such as the alpha4beta1 integrin and CD36, exclusively present on stress reticulocytes, and CD47 on mature RBCs. More recently, attention focused on Lu/BCAM, the unique RBC receptor for laminin, and on ICAM-4, a red cell-specific adhesion receptor, which is a ligand for a large repertoire of integrins (alphaLbeta2, alphaMbeta2, alphaxbeta2, alphaVbeta3). The counter-receptors on ECs and the role of plasma proteins forming bridges between blood cells and ECs have been clarified in part. It has also been shown that reticulocytes from SCD patients express higher levels of alpha4beta1 integrin and CD36, and that under hydroxyurea (HU) therapy, both cell adhesion to ECs or extracellular matrix proteins and the levels of these adhesion molecules are reduced. These findings are consistent with the view that enhanced adhesion of blood cells to ECs is largely determined by the membrane expression level of adhesion molecules and could be a crucial factor for triggering or aggravating vaso-occlusion. In SCD patients, membrane expression of Lu/BCAM (and perhaps ICAM-4) is enhanced on RBCs whose adherence to laminin or ECs is also increased. Interestingly, Lu/BCAM- and ICAM-4-mediated adhesion are enhanced by the stress mediator epinephrine through a PKA-dependent pathway initiated by a rise in intracellular cAMP and leading to receptor activation by phosphorylation according to the same signaling pathway. More recently, studies based on quantitative expression analysis of adhesion molecules on RBCs and during erythroid differentiation in patients undergoing HU therapy, surprisingly revealed that Lu/BCAM level was enhanced, although alpha4beta1, CD36 and ICAM-4 (to a lower extent) levels were indeed reduced. CD47 and CD147 expression were also enhanced in HU-treated patients. Based on these findings we suggest that the signalization cascade leading to receptor activation rather than the expression level only of adhesion molecules may be the critical factor regulating cell adhesion, although both mechanisms are not mutually exclusive.     

Detection of a LFA-1-like epitope on the surface of erythrocytes infected with a strain of Plasmodium falciparum.

The adhesion of erythrocytes infected with Plasmodium falciparum (P. falciparum) is one of the major pathological features of severe malaria. Several potential receptors to endothelium for falciparum-infected erythrocyte on endothelium have been described. Recently, the malaria binding site on ICAM-1(CD54) has been mapped to a site distinct but overlapping with the LFA-1 (CD11a/CD18) site. We detected by flow cytometry, confocal laser microscopy and immunoprecipitation, a molecule expressed at the surface of erythrocytes infected with mature stages of the M96 strain of P. falciparum that was recognized by a monoclonal antibody (mAb) (TS1/22) directed against an LFA-1 epitope. However, this molecule was not recognized by mAbs directed against other epitopes of LFA-1 or against other integrins. Furthermore, the mAb TS1/22 partially inhibited cytoadherence of parasitized red blood cells to human-brain microvascular endothelial cells. The expression of a molecule sharing an epitope with human LFA-1 integrin on the parasitized erythrocyte surface could be involved in the sequestration of these cells and thus in the pathogenesis of severe disease.     

Calcium homeostasis of human erythrocytes and its pathophysiological implications

In human red cells, Ca is mainly bound to the inner side of the plasma membrane. A smaller part may be present within intracellular Ca storing vesicles, while only a few percent of total red cell Ca is in ionized form. In some hemolytic anemias (sickle cell anemia,-thalassemia), an increased number of endocytotic vesicles storing Ca is probably responsible for the elevation of total red cell Ca content. Red cell Ca inward transport, which is partially susceptible to inhibition by Ca entry blockers, has been reported to be enhanced by physiological shear stress and enrichment in membrane cholesterol, as well as in some hemolytic anemias. Normal intracellular ionized Ca levels have been assessed in several diseases where elevated Ca inward transport rates or decreased Ca efflux through the Ca pump (hemolytic anemias, cystic fibrosis, essential hypertension) had been observed previously. Thus, red cell Ca homeostasis is apparently capable of keeping ionized Ca levels within the physiological range of 20–60 nM under most pathological conditions investigated so far.Conceptually, changes in red cell Ca homeostasis (or also in other red cell membrane parameters) may be of pathophysiological importance in two respects:

Study of irreversibly sickled cells in an animal model

Erythrocytes (RBCs) from six patients with sickle cell anemia were transfused to laboratory rats in order to study the intravascular survival of irreversibly sickled cells (ISCs). Fifteen minutes after transfusion, a mean of 48.8 percent (range 23-95 percent) of the ICSs injected were present in the rats'' blood, a value that was significantly lower than that for the total population of sickle cell anemia erythrocytes transfused (mean 82.4 percent, range 36-114 percent). The intravascular half-life of ISCs was also lower (mean 0.83 hours ± 0.18 SD) than that observed for the total sickle cell anemia erythrocytes (mean 1.62 hours ± 0.19 SD) during the initial two hours of the transfusion experiments. The irreversibly sickled cells that remained in the rats'' blood thereafter survived as well as those cells that were not irreversibly sickled. Severe hypoxia in the recipient animals did not appear to selectively remove ISCs from circulation. These data are consistent with heterogeneity of ISCs in terms of their intravascular viability. Some ISCs may have adapted to the stress of circulation despite their abnormal shape.     

Biostructures en environnement électromagnétique

Les interactions ondes électromagnétiques/biostructures peuvent être recherchées et, en ce sens, générées volontairement lors d'une thérapie ou d'un diagnostic. Toutes les études menées tendent à répondre à la question de l'existence ou non d'un risque clairement identifié pour l'être humain. Nous tentons dans cet article de classer les paramètres mis en jeu afin d'en dégager les informations nécessaires à toute étude, en nous limitant au cas des fréquences intermédiaires (3 kHz-3 MHz). L'étude des effets d'un environnement électromagnétique sur les dispositifs biomédicaux est illustrée par l'exemple des stimulateurs cardiaques.     

Un filtre numérique basé sur la dérivation non-entière pour l'analyse du signal électrocardiographique

Cet article présente une méthode de synthèse basée sur la dérivation non-entière d'un filtre passe-bande de type filtre à réponse impulsionnelle finie (RIF) pour l'analyse du signal électrocardiographique. Un modèle fractal de la densité spectrale de puissance du complexe QRS est utilisé pour déterminer l'ordre non-entier négatif du premier filtre qui maximise le rapport signal sur bruit. Le bruit résiduel de basse fréquence est réduit par un deuxième filtre avec un ordre non-entier positif. Les deux filtres, dont les coefficients ne dépendent que des ordres non-entiers, sont mis en cascade pour former un filtre passe-bande. Des applications aux signaux ECG du Massachussets Institute of Technology (MIT)/BIH arhythmia database sont présentées et illustrent les capacités de cet algorithme à mettre en évidence les complexes QRS d'ECG fortement bruité.