Molecular Research of Regulation of Red Blood Cells in Health, Hereditary or Acquired Diseases.

During the first era of humanity, the conditions of life, including hunting, fighting, obtaining food, and diseases, were associated with frequent hemorrhages, anemia, and infections, which led to death or untreatable conditions [...].

Pro- and Anti-Inflammatory Prostaglandins and Cytokines in Humans: A Mini Review.

Inflammation has been described for two millennia, but cellular aspects and the paradigm involving different mediators have been identified in the recent century. Two main groups of molecules, the prostaglandins (PG) and the cytokines, have been discovered and play a major role in inflammatory processes. The activation of prostaglandins PGE2, PGD2 and PGI2 results in prominent symptoms during cardiovascular and rheumatoid diseases. The balance between pro- and anti-inflammatory compounds is nowadays a challenge for more targeted therapeutic approaches. The first cytokine was described more than a century ago and is now a part of different families of cytokines (38 interleukins), including the IL-1 and IL-6 families and TNF and TGFß families. Cytokines can perform a dual role, being growth promotors or inhibitors and having pro- and anti-inflammatory properties. The complex interactions between cytokines, vascular cells and immune cells are responsible for dramatic conditions and lead to the concept of cytokine storm observed during sepsis, multi-organ failure and, recently, in some cases of COVID-19 infection. Cytokines such as interferon and hematopoietic growth factor have been used as therapy. Alternatively, the inhibition of cytokine functions has been largely developed using anti-interleukin or anti-TNF monoclonal antibodies in the treatment of sepsis or chronic inflammation.

Vascular Permeability in Diseases.

Vascular permeability is a selective mechanism that maintains the exchange between vessels, tissues, and organs. The regulation was mostly studied during the nineteenth century by physiologists who defined physical laws and equations, taking blood, tissue interstitial, and oncotic pressure into account. During the last decades, a better knowledge of vascular cell functions and blood-vessel interactions opens a new area of vascular biology. Endothelial cell receptors vascular cell adhesion molecule (VCAM), intercellular cell adhesion molecule (ICAM), vascular endothelial growth factor receptor (VEGFR-2), receptor for advanced glycation end products (RAGE), and mediators were identified and their role in homeostasis and pathological situations was described. The molecular differences of endothelial cell junctions (tight, gap, and adherens junctions) and their role in vascular permeability were characterized in different organs. The main mediators of vasomotricity and permeability, such as prostaglandins, nitric oxide (NO), prostacyclin, vascular growth factor (VEGF), and cytokines, have been demonstrated to possess major functions in steady state and pathological situations. Leukocytes were shown to adhere to endothelium and migrate during inflammatory situations and infectious diseases. Increased vascular permeability is linked to endothelium integrity. Glycocalyx, when intact, may limit cancer cell metastasis. Biological modifications of blood and tissue constituents occurring in diabetes mellitus were responsible for increased permeability and, consequently, ocular and renal complications. Vascular pressure and fluidity are major determinants of pulmonary and cerebral edema. Beside the treatment of the infectious disease, of the blood circulation dysfunction and inflammatory condition, drugs (cyclooxygenase inhibitors) and specific antibodies anti-cytokine (anti-VEGF) have been demonstrated to reduce the severity and the mortality in diseases that exhibited enhanced vascular permeability.

Old and New Blood Markers in Human Colorectal Cancer.

Cancer is a predominant cause of mortality all over the world. Lung, prostate, and colorectal cancer are the more frequent in men while breast and colorectal have a high incidence in women. Major progress aside, some cancers are still frequent and one major issue is improvements in detection methods. Imaging techniques have a major role, but inflammatory, tumoral markers and calculated scores may contribute to the assessment of prognosis. The erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and carcinoembryonic antigen cell adhesion molecule (CEACAM) have been used for decades and do not have a clear use for diagnosis or prognosis yet. The CEACAM family includes 12 human members, and some of them have a cluster differentiation (CD). CD66 may be an interesting indicator of disease severity. Beside interleukin-6 (IL-6), the high level of which is observed in patients with a high mortality rate, other cytokines IL-17A, IL-22, and transforming growth factor -ß (TGF-ß) are expressed at the tumor level. The detection of circulating tumor cells has been improved but is still of undetermined value. Circulating tumor DNA (ctDNA) was recently studied in CRC stage II patients and may be helpful for chemotherapy management.

Endothelial Cell Participation in Inflammatory Reaction.

Inflammation is an old concept that has started to be considered as an important factor in infection and chronic diseases. The role of leukocytes, the plasmatic components, then of the mediators such as prostaglandins, cytokines, and, in recent decades, of the endothelium has completed the concept of the inflammation process. The function of the endothelium appeared to be crucial as a regulator or the initiator of the inflammatory process. Culture of human endothelial cells and experimental systems made it possible to define the molecular basis of inflammation in vascular diseases, in diabetes mellitus, atherosclerosis, vasculitis and thromboembolic complications. Advanced glycation end product receptor (RAGE), present on endothelial cells (ECs) and monocytes, participates in the activation of these cells in inflammatory conditions. Inflammasome is a cytosolic multiprotein that controls the response to diverse microorganisms. It is positively regulated by stimulator of interferon response CGAMP interactor-1 (STING1). Angiogenesis and thrombotic events are dysregulated during inflammation. ECs appear to be a protector, but also a possible initiator of thrombosis.

Cellular and Molecular Aspects of Blood Cell-Endothelium Interactions in Vascular Disorders.

In physiology and pathophysiology the molecules involved in blood cell-blood cell and blood cell-endothelium interactions have been identified. Platelet aggregation and adhesion to the walls belonging to vessels involve glycoproteins (GP), GP llb and GP llla and the GP Ib-IX-V complex. Red blood cells (RBCs) in normal situations have little interaction with the endothelium. Abnormal adhesion of RBCs was first observed in sickle cell anemia involving vascular cell adhesion molecule (VCAM)-1, α4ß1, Lu/BCAM, and intercellular adhesion molecule (ICAM)-4. More recently RBC adhesion was found to be increased in retinal-vein occlusion (RVO) and in polycythemia vera (PV). The molecules which participate in this process are phosphatidylserine and annexin V in RVO, and phosphorylated Lu/BCAM and α5 laminin chain in PV. The additional adhesion in diabetes mellitus occurs due to the glycated RBC band 3 and the advanced glycation end-product receptors. The multiligand receptor binds advanced glycation end products (AGEs) or S100 calgranulins, or ß-amyloid peptide. This receptor for advanced glycation end products is known as RAGE. The binding to RAGE-activated endothelial cells leads to an inflammatory reaction and a prothrombotic state via NADPH activation and altered gene expression. RAGE blockade is a potential target for drugs preventing the deleterious consequences of RAGE activation.

JAK2V617F activates Lu/BCAM-mediated red cell adhesion in polycythemia vera through an EpoR-independent Rap1/Akt pathway.

Polycythemia vera (PV) is characterized by an increased RBC mass, spontaneous erythroid colony formation, and the JAK2V617F mutation. PV is associated with a high risk of mesenteric and cerebral thrombosis. PV RBC adhesion to endothelial laminin is increased and mediated by phosphorylated erythroid Lu/BCAM. In the present work, we investigated the mechanism responsible for Lu/BCAM phosphorylation in the presence of JAK2V617F using HEL and BaF3 cell lines as well as RBCs from patients with PV. High levels of Rap1-GTP were found in HEL and BaF3 cells expressing JAK2V617F compared with BaF3 cells with wild-type JAK2. This finding was associated with increased Akt activity, Lu/BCAM phosphorylation, and cell adhesion to laminin that were inhibited by the dominant-negative Rap1S17N or by the specific Rap1 inhibitor GGTI-298. Surprisingly, knocking-down EpoR in HEL cells did not alter Akt activity or cell adhesion to laminin. Our findings reveal a novel EpoR-independent Rap1/Akt signaling pathway that is activated by JAK2V617F in circulating PV RBCs and responsible for Lu/BCAM activation. This new characteristic of JAK2V617F could play a critical role in initiating abnormal interactions among circulating and endothelial cells in patients with PV.

Red blood cell adhesion in diabetes mellitus is mediated by advanced glycation end product receptor and is modulated by nitric oxide.

Red blood cell (RBC) adhesion to endothelium is increased in diabetes mellitus and is correlated with the severity of vascular complications. Microangiopathy is the most frequent complications in patients suffering from diabetes mellitus. Elevated glucose concentration increases the oxidation phenomenon and advanced glycation end product (AGE) formation. Plasma proteins, structural proteins and also RBC proteins can be glycated such as glycated hemoglobin and RBC membrane proteins. Interaction of plasmatic AGE or RBC bearing AGE with the receptor for AGE (RAGE) alters vascular function leading to a vascular hyperpermeability inflammatory reaction including oxidant stress and cytokine production. Reactive oxygen species (ROS) react with nitric oxide (NO) limiting its vasodilatory effect and NO synthase function is altered. All these factors may be at the origin of high blood pressure which is deleterious for the eye and kidney vasculature. AGE can act directly on vascular function but also through RAGE. AGE binding to RAGE alters endothelial cell function stimulating NADPH oxidase and reactive oxygen species production. Limiting oxidation, reducing AGE formation or interaction with RAGE is achievable by drugs already used for hypertension or diabetes, but new treatment by NO modulators may limit the deleterious effect of RBC adhesion to endothelium.

Activation of the receptor for advanced glycation end products and consequences on health.

Advanced glycation end products (AGE) resulted from a reaction between free amino group of proteins and carbohydrates. This reaction is followed by oxidation and molecular rearrangement. Alternatively AGEs can be produced by glycolysis and oxidation. AGEs bind to a cellular receptor RAGE. RAGE engagement by ligands AGE, ß-amyloid peptide, and S100 calgranulin induces a stimulation of NADPH oxidase, reactive oxygen intermediate formation, NFκB activation and gene transcription. This cascade of reaction leads to an inflammatory reaction responsible for alteration of microvessels in the retina and the kidney. Blockade of RAGE by antibodies anti-RAGE, TTP488 (azeliragon), or rRAGE prevents or limits the deleterious effect of AGEs.

Protein glycation: a firm link to endothelial cell dysfunction.

The advanced glycation end products (AGEs) are a heterogeneous class of molecules, including the following main subgroups: bis(lysyl)imidazolium cross-links, hydroimidazolones, 3-deoxyglucosone derivatives, and monolysyl adducts. AGEs are increased in diabetes, renal failure, and aging. Microvascular lesions correlate with the accumulation of AGEs, as demonstrated in diabetic retinopathy or renal glomerulosclerosis. On endothelial cells, ligation of receptor for AGE (RAGE) by AGEs induces the expression of cell adhesion molecules, tissue factor, cytokines such as interleukin-6, and monocyte chemoattractant protein-1. A chief means by which AGEs via RAGE exert their effects is by generation of reactive oxygen species, at least in part via stimulation of NADPH oxidase. Diabetes-associated vascular dysfunction in vivo can be prevented by blockade of RAGE. Thus, agents that limit AGE formation, increase the catabolism of these species, or antagonize their binding to RAGE may provide new targets for vascular protection in diabetes.

Changes in glycation and oxidation markers in patients starting peritoneal dialysis: a pilot study.

BACKGROUND: The high incidence of cardiovascular disease in uremic patients makes it a major cause of morbidity and mortality in those patients. Uremia is associated with carbonyl and oxidative stress, which result in the enhanced formation of glycation and oxidation products respectively. In the present study, the blood levels of advanced glycation end products (AGEs) and advanced oxidation protein products (AOPPs) were investigated in uremic patients prior to and after initiation of peritoneal dialysis (PD). METHODS: 22 patients [11 nondiabetic (G1) and 11 diabetic (G2) subjects] were enrolled in a single-center prospective study. Prior to starting PD (TO) and 6 and 12 months later, changes in AGE and AOPP levels were analyzed in the total study population and in each group (Friedman test, intragroup). At each time point, a comparison was made between the levels of the above-mentioned products in G1 and G2 (Mann-Whitney test, intergroup). Correlations between AGE or AOPP levels and residual renal function, peritoneal creatinine clearance, glucose peritoneal equilibration test, or daily dextrose exposure were analyzed using the Pearson test. RESULTS: At TO, no significant difference was found between the two groups for AGE or AOPP levels. Initiation of PD was followed by an increase in AGE levels in all patients (p < 0.01 at 6 and 12 months). AGE Levels were higher in G2 than in G1 at 12 months after the start of PD (p < 0.05). In contrast to G2 results, initiation of PD in G1 led to reduced AOPP Levels (at 6 and 12 months, p = 0.01 and p < 0.05 respectively). However, no correlation between AGE or AOPP levels and residual renal function, peritoneal creatinine clearance, glucose peritoneal equilibration test, or daily dextrose exposure could be established. CONCLUSION: This study demonstrates that PD is associated with an increase in levels of blood glycation end products, particularly in diabetic patients, but also with a decrease in oxidative products such as AOPPs, especially in nondiabetic subjects.

Modulation of RAGE expression influences the adhesion of red blood cells from diabetic patients.

Atherothrombotic complications are frequent in patients with type 2 diabetes. Red blood cells (RBC) from diabetic patients exhibited an increased adhesion which correlated to the extent of vascular complications. In the present study we have investigated the adhesive interactions of RBCs with endothelium, using flow-based assessments. RBCs and endothelial cells were unstimulated or stimulated using respectively adrenaline and TNFalpha. Adhesion assays were carried-out by drawing the RBC suspension through a glass microcapillary tube precoated by human umbilical vein endothelial cells. These microslides were then incorporated into a controlled flow system equipped with a computerized video-microscopic image analysis. RBCs from diabetic patients bind to endothelial cells and could withstand wall shear stresses above 0.1 Pa. After stimulation by TNFalpha the adhesion was 1.5-fold higher. Blocking experiments demonstrated that the adhesion was mediated by the receptor for AGE (RAGE). Adrenaline-treated RBCs showed a transient increase in adhesion at low shear stresses. Inflammatory mediators or catecholamine amplifying diabetic RBC adhesion may aggravate endothelial cell damages.

[Glycation, glycoxidation and diabetes mellitus]. / Glycation, glycoxydation et diabète.

Advanced glycation end-products (AGEs) result from a reaction between carbohydrates and the free amino groups of proteins, lipids, and DNA. Non enzymatic glycation, glycoxidation with glucose auto-oxidation and the polyol pathway are involved in glycated protein formation. AGEs also named glycotoxins are found in excess in pathological situations such as diabetes mellitus, renal failure, and aging or after absorption of food containing glycated products. Three major pathophysiological mechanisms are described to explain AGE toxicity, first AGEs can accumulate in the vessel wall and in collagen of different tissues; second in situ glycation is possible; third, AGEs bind to cell receptors inducing deleterious consequences. AGE receptor RAGE is a multiligand member of the immunoglobulin superfamily of cell surface molecules. AGE-receptor interaction can alter, macrophage, endothelial cell, mesangial and mesothelial cell functions and can induce inflammation. Oxidant stress, vascular hyperpermeability, vascular cell adhesion molecule-1 (VCAM-1) overexpression and monocytes chemotactic Protein-1 (MCP-1) production have been observed after cell activation by AGEs. AGEs appear to be involved in the genesis of diabetic macro but also microangiopathy such as retinopathy and glomerulosclerosis. New drugs are tested to prevent or break the AGE-protein cross-linkage, or to control the AGE-receptor interaction and their consequences. Dietary treatment, strict glycemic control and preservation of renal function remain the best approach for preventing AGE formation and limiting their deleterious effects.

[Biocompatibility of peritoneal dialysis fluids]. / La biocompatibilité des solutions de dialyse péritonéale.

Repeated and long-term exposure to conventional glucose-based peritoneal dialysis fluids (PDFs) with poor biocompatibility plays a central role in the pathogenesis of the functional and structural changes of the peritoneal membrane. We have used immortalized human peritoneal mesothelial cells in culture to assess in vitro the biocompatibility of PDFs. Low pH, high glucose concentration and heat sterilization represent major factors of low biocompatibility. Two recent groups of glucose derivatives have been described. Glucose degradation products (GDPs) are formed during heat sterilization (glycoxidation) and storage. GDPs can bind protein and form AGEs (Advanced Glycation End-products), which can also result from the binding of glucose to free NH2 residues of proteins (glycation). The physiological pH, and the separation of glucose during heat sterilization (low GDP content) in the most recent PDFs dramatically increase the biocompatibility. The choice of PD programs with high biocompatibility PDFs allows preserving the function of the peritoneal membrane. Improvement of PDF biocompatibility may limit the occurrence of chronic chemical peritonitis and may allow long-term PD treatment.

AGEs, macrophage colony stimulating factor and vascular adhesion molecule blood levels are increased in patients with diabetic microangiopathy.

In vitro experiments and animal models indicate that advanced glycation end products (AGEs) may play a crucial role in the vascular dysfunctions observed in patients with diabetes mellitus. These results prompted us to study subrogate markers of inflammation or vascular dysfunction in type II diabetic patients. Monocyte count and activation are dependent upon macrophage colony stimulating factors (M-CSF). Soluble vascular cell adhesion molecule (sVCAM-1) blood levels have been proposed as a marker for endothelium activation. To explore a possible relationship between these factors in diabetic patients, we measured a chemically defined AGE, N(carboxymethyl)lysine-protein (CML-protein) in a group of normal subjects (n = 55) and of diabetic patients (n = 40) using ELISA. Simultaneously, we determined M-CSF and sVCAM-1 blood levels. We found that CML-protein blood levels were significantly higher in patients with diabetes compared to non-diabetic subjects (40.2 +/- 4.7 and 7.9 +/- 0.7 pmol/mg protein respectively, p < 0.0001). M-CSF was increased while sVCAM-1 blood levels were normal in the group of diabetics. M-CSF blood level was correlated to CML-protein blood level (p < 0.05). In addition CML-protein, M-CSF and sVCAM-1 were increased in patients with micro-angiopathy. These results suggest that AGE may contribute to vascular dysfunction including microangiopathy.

Erythrocytes and platelet adhesion to endothelium are mediated by specialized molecules.

Erythrocytes in normal conditions have weak interactions with other blood cells and endothelial cells while in pathological circumstances they can adhere to endothelium and aggregate or agglutinate to blood cells. Erythrocyte adhesion was found to be abnormal in sickle cell anemia and diabetes mellitus and correlated to the vascular complications. Further studies demonstrated that VLA-4 adhesion molecule (alpha4beta1) present on erythrocytes bound to vascular cell adhesion molecule (VCAM-1) of the endothelium. In addition, the blood group Lutheran molecule (LU) overexpressed on sickle erythrocytes bind to laminin present on cells or in the intercellular space. In diabetes mellitus the formation of advanced glycation end products (AGE) by reaction between carbohydrates and free aminogroups of lysine is responsible for red blood cell membrane glycation. AGEs present on RBCs bind to the receptor for AGE (RAGE) on endothelium, activating endothelial cells. A molecule related to blood group Rhesus was demonstrated to belong to the intercellular adhesion molecule (ICAM) family. ICAM-4 binds to integrins present on leukocytes (CD11-CD18) and on platelets (alpha2beta4) offering a surface, which can be involved in thrombosis. The identification of erythrocytes adhesion molecules open a new way to understand thrombotic processes and vascular dysfunction.

Molecular basis of erythrocyte adhesion to endothelial cells in diseases.

Red blood cell (RBC) adhesion to endothelium can be studied in static and flow conditions. Increased RBC adhesion was first described in sickle cell disease. Several molecules were shown to be involved in this phenomenon: VCAM-1, α4ß1, Lu/BCAM, ICAM-4. In malaria, Plasmodium falciparum erythrocyte membrane protein1 binds to ICAM-1, PECAM-1 and facilitates the parasite dissemination. In diabetes mellitus augmented RBC adhesion is correlated to the severity of vascular complications. Glycated RBC band3 reacts with the endothelial Receptor for Advanced Glycation End products (RAGE). RAGE engagement induced endothelial cell dysfunction. In patients with Polycythemia Vera (PV), the most frequent myeloproliferative disorder, constitutive phosphorylation of RBC Lu/BCAM is responsible for an increased adhesion to endothelial cell laminin. Retinal vein occlusion (RVO) is a common cause of permanent visual loss. Spontaneous growth of erythroid precursors was observed in more than 25% of patients. RBC adhesion was enhanced and correlated to phosphatidyl serine (PS) expression on RBC. Anti-PS receptor blocked RVO RBC adhesion indicating that the counterpart of RBC PS is PS endothelial cell receptor. Erythrocyte adhesion is mediated by different molecule abnormalities in different diseases but is associated to a higher risk of thrombosis and vascular complications.