Rubbing salt into wounded endothelium: sodium potentiates proatherogenic effects of TNF-α under non-uniform shear stress.

Increased consumption of sodium is a risk factor for hypertension and cardiovascular diseases. In vivo studies indicated that high dietary sodium may have a direct negative influence on endothelium. We investigated the effects of high sodium on the endothelial activation during early steps of atherogenesis. Endothelial cells (HUVECs) grown in a model of arterial bifurcations were exposed to shear stress in the presence of normal or high (+ 30 mmol/l) sodium. Adherent THP-1 cells, and the adhesion molecule expression were quantified. Sodium channel blockers, pathways' inhibitors, and siRNA against tonicity-responsive enhancer binding protein (TonEBP) were used to identify the mechanisms of sodium effects on endothelium. ApoE-deficient mice on low-fat diet received water containing normal or high salt (8% w/v) for four weeks, and the influence of dietary salt on inflammatory cell adhesion in the common carotid artery and carotid bifurcation was measured by intravital microscopy. In vitro, high sodium dramatically increased the endothelial responsiveness to tumour necrosis factor-α under non-uniform shear stress. Sodium-induced increase in monocytic cell adhesion was mediated by reactive oxygen species and the endothelial nitric oxygen synthase, and was sensitive to the knockdown of TonEBP. The results were subsequently confirmed in the ApoE-deficient mice. As compared with normal-salt group, high-salt intake significantly enhanced the adhesion of circulating CD11b+ cells to carotid bifurcations, but not to the straight segment of common carotid artery. In conclusion, elevated sodium has a direct effect on endothelial activation under atherogenic shear stress in vitro and in vivo, and promotes the endothelial-leukocyte interactions even in the absence of increased lipid concentrations.

CD40/CD154 system and pro-inflammatory cytokines in young healthy male smokers without additional risk factors for atherosclerosis.

OBJECTIVE AND DESIGN: Atherosclerosis, as an inflammatory disease, is characterized by pathologically altered levels of cytokines. We investigated whether smoking affects the CD40/CD154 system and pro-inflammatory cytokines in young males without other risk factors for atherosclerosis. SUBJECTS: Young male smokers (n=13) and 14 non-smoking controls were investigated. METHODS: The differences in CD40/CD154 system and serum cytokines between the groups were measured using flow cytometry and ELISA. RESULTS: In smokers, there was a strong trend (P<0.06) for increased CD40 expression on platelets as compared with non-smokers. However, there were no significant differences in CD40 expression on monocytes or in CD154 expression on platelets and T-cells between smokers and non-smokers. There was a strong trend for increased platelet-monocyte aggregates in smokers (P<0.06). Also, smokers had slightly but not significantly elevated hsCRP and IL-6 levels, and slightly decreased TNF-alpha and MCP-1. Interestingly, IL-18, a cytokine which has the ability to promote both Th1 and Th2 responses, was significantly decreased in smokers group (P=0.03 vs controls). CONCLUSIONS: In young healthy males, smoking is not associated with dramatic changes in CD40/CD154 system. However, cigarette smoke alters the secreted cytokine profile, leading to significant decrease in systemic IL-18 levels.

Increased prolactin in acute coronary syndromes as putative Co-activator of ADP-stimulated P-selectin expression.

Prolactin and leptin are newly recognized platelet co-stimulators due to enhancement of ADP-induced platelet aggregation. The aim of our study was to assess whether both hormones prolactin and leptin play a role as co-activators of platelet activation in patients with acute coronary syndromes. Twenty-one patients with acute coronary syndromes, 10 with stable angina pectoris and 10 controls were studied. Patients with acute coronary syndromes showed significantly higher prolactin and leptin values and a significant increased P-selectin expression on platelets compared to patients with stable angina pectoris or controls. However, patients with acute myocardial infarction as a subgroup of acute coronary syndromes showed the highest prolactin levels as well as ADP stimulated P-selectin expression. In the myocardial infarction subgroup prolactin values showed a significant correlation to ADP stimulated P-selectin expression on platelets (r (2)=0.41; p=0.025), whereas leptin was not correlated. Our data indicate an association between increased prolactin values and enhanced P-selectin expression on platelets in patients with acute coronary syndromes. Therefore, the stress hormone prolactin could be a co-stimulator of platelet activation in these patients. In contrast, the putative platelet activator leptin does not seem to play a major role in acute coronary syndromes.

Contribution of Src-FAK signaling to the induction of connective tissue growth factor in renal fibroblasts.

Expression of connective tissue growth factor (CTGF) is sensitive to reorganization of the actin cytoskeleton, but also to alterations in cell morphology due to extracellular forces, for example, cyclic stretching or mechanical loading. Dynamic alterations of focal adhesion proteins were thus proposed to modulate CTGF induction. Immortalized human renal fibroblasts were cultured in or on top of preformed collagen-1 gels. Proteins were detected by immunofluorescence and quantified by Western blotting. Fibroblasts cultured in/on collagen gels resembled cells in vivo by their spindle-like morphology, absence of actin stress fibers, small punctiform focal contacts, and low levels of CTGF expression. Disassembly of microtubules by short-term treatment with colchicine induced cell rounding, cortical recruitment of patchy F-actin, reorganization of focal contacts into strong clusters, and upregulation of CTGF, all of which were dependent on RhoA-Rho-kinase signaling. Clustering of focal adhesion sites activated Src-family kinases and focal adhesion kinase (FAK). Interference with Src activity by PP2 had no effect on the morphological alterations but decreased tyrosine phosphorylation of focal adhesion proteins and almost completely prevented upregulation of CTGF. Furthermore, inhibition of phosphatidylinositol 3-kinase reduced CTGF expression. On the other hand, when the fibroblasts were cultured on a rigid matrix, that is collagen-coated plates, strong focal complexes prevented the dynamic alterations, and RhoA-mediated upregulation of CTGF expression was independent of Src-FAK signaling. Assembly of focal adhesion proteins regulates CTGF expression, providing a link between actin network, adhesion receptors, and CTGF-mediated functions such as synthesis of extracellular matrix proteins.

Effects of dietary triglycerides on rheological properties of human red blood cells (abstract).

Atherogenic diets rich in saturated fat and cholesterol influence the blood viscosity and red blood cell (RBC) aggregability, the parameters associated with increased risk of circulatory disorders. However, little is known about the effect of triglycerides, which are the major dietary lipid form in humans, on blood rheology. Therefore, we studied the effects of postprandial plasma triglyceride levels on human RBC indices, hematological parameters, RBCs aggregation velocity and whole blood viscosity. For this purpose, whole blood was collected 2 hours after high-fat or low-fat meal. Proteins, triglycerides and cholesterol levels of plasma were analysed, and RBCs rouleaux formation rate was measured in 70% autologous plasma using a low-shear rheoscope. There were no significant differences in hematological parameters, RBC indices, whole blood viscosity, plasma protein and cholesterol content between high-fat and low-fat blood samples. However, a significant increase in rouleaux formation rate was observed in samples with high postprandial triglyceride levels, when compared with low-triglyceride samples. Plasma triglyceride levels correlated significantly with rouleaux formation rate. In conclusion, these results suggest that diet-dependent alterations of plasma triglyceride levels as well as possible changes in the cell membrane lipid composition lead to RBC hyperaggregability.

Delay of neutrophil apoptosis in acute coronary syndromes.

Apoptosis of polymorphonuclear neutrophils (PMN) is currently discussed as a key event in the control of inflammation. This study determined PMN apoptosis and its underlying mechanisms in controls (C), patients with stable (SAP) or unstable angina (UAP), and with acute myocardial infarction (AMI). Blood was drawn from 15 subjects of each C, SAP, UAP, and AMI. Apoptosis was measured by flow cytometry in isolated PMN (propidium iodide staining) and PMN from whole blood (CD16, FcgammaRIII). Serum cytokines were determined by enzyme-linked immunosorbent assay. Apoptosis of isolated PMN was delayed significantly in acute coronary syndromes (ACS) as compared with SAP or C (C, 51.2+/-12.6%; SAP, 44.9+/-13.6%; UAP, 28.4+/-10.1%; AMI, 20.3+/-8.5%; AMI or UAP vs. SAP or C, P<0.001). These results were confirmed by measurement of PMN apoptosis in cultured whole blood from patients and controls. Moreover, serum of patients with ACS markedly reduced apoptosis of PMN from healthy donors. Analysis of patients' sera revealed significantly elevated concentrations of tumor necrosis factor alpha, interferon-gamma (IFN-gamma), granulocyte macrophage-colony stimulating factor (GM-CSF), and interleukin (IL)-1beta in ACS (vs. C and SAP). IFN-gamma, GM-CSF, and IL-1beta significantly delayed PMN apoptosis in vitro. Furthermore, coincubation of PMN with adenosine 5'-diphosphate-activated platelets significantly inhibited PMN apoptosis as compared with coculture with unstimulated platelets. This study demonstrates a pronounced delay of PMN apoptosis in UAP and AMI, which may result from increased serum levels of IFN-gamma, GM-CSF, and IL-1beta and from enhanced platelet activation. Therapeutical modulation of these determinants of PMN lifespan may provide a new concept for the control of inflammation in ACS.

Enhancement of red blood cell aggregation by plasma triglycerides.

The effects of plasma triglycerides level on human red blood cells (RBCs) indices, hematological parameters, RBCs aggregation velocity and whole blood viscosity were studied at 2 hours after high-fat or low-fat meal. Proteins, triglycerides and cholesterol levels of plasma were analysed. The RBCs rouleaux formation rate was measured in 70% autologous plasma (with 30% phosphate-buffered saline, PBS) or 1 g/dl dextran T70 solution (with 4 g/dl bovine serum albumin) in PBS, using a low-shear rheoscope. The results were grouped according to triglycerides content in plasma. No significant difference in whole blood viscosity, hematological parameters, RBC indices, protein and cholesterol content was observed between high-fat and low-fat blood samples. There was a significant increase in rouleaux formation rate of samples with high triglyceride levels, when measured in 70% autologous plasma, but it was not significant in dextran T70 containing medium. In conclusion, the results obtained suggest that alteration of plasma lipid levels as well as possible changes in the cell membrane lipid composition lead to enhanced RBC aggregation.

O(2) release from erythrocytes flowing in a narrow O(2)-permeable tube: effects of erythrocyte aggregation.

The effects of erythrocyte aggregation on O(2) release were examined using O(2)-permeable fluorinated ethylenepropylene copolymer tubes (inner diameter, 25 microm; outer diameter, 100 microm). Measurements were performed using an apparatus built on an inverted microscope that contained a scanning-grating spectrophotometer with a photon count detector connected to two photomultipliers and an image processor through a video camera. The rate of O(2) release from the cells flowing in the narrow tube was determined based on the visible absorption spectrum and the flow velocity of the cells as well as the tube size. When the tube was exposed to nitrogen-saturated deoxygenated saline containing 10 mM sodium dithionite, the flowing erythrocytes were deoxygenated in proportion to the traveling distance, and the deoxygenation at a given distance increased with decreasing flow velocity and cell concentration (hematocrit). Adding Dextran T-70 to the cell suspension increased erythrocyte aggregation in the tube, which resulted in suppressed cell deoxygenation and increased marginal cell-free-layer thickness. The deoxygenation was inversely proportional to the cell-free-layer thickness. The relation was not essentially altered even when the medium viscosity was adjusted with Dextran T-40 to remain constant. The rate of O(2) release from erythrocytes in the tube was discussed in relation to the O(2) diffusion process. We conclude that the diffusion of O(2) from erythrocytes flowing in narrow tubes is inhibited primarily by erythrocyte aggregation itself and partly by thickening of the cell-free layer.

Aggregation and sedimentation of mixtures of erythrocytes with different properties.

The rouleau formation of erythrocytes and the erythrocyte sedimentation were examined for mixture of different kinds of the cells suspended in isotonic phosphate-buffered saline containing 1 or 2 g/dl dextran T-70 (MW = 70,400) and 4 g/dl albumin, using a low shear rheoscope and the Westergren method, respectively. The deformability of cells was decreased by treating with diamide, diazene-dicarboxylic acid bis[N,N-dimethylamide], and the sialic acid content of cells, i.e., the surface negative charge, was reduced by treating with neuraminidase. (1) The rate of rouleau formation was decreased in cells with decreased deformability, while it was increased in cells with reduced sialic acid content. The rate changed in proportion to the ratio of the modified cells to normal cells. (2) The erythrocyte sedimentation was also decreased in cells with decreased deformability, while it was increased in cells with reduced sialic acid content. Furthermore, the erythrocyte sedimentation changed almost proportionally to the ratio of the modified cells to normal cells. (3) When normal deformable cells were mixed with cells with decreased deformability, the deformable cells seemed to settle faster than the less deformable cells, though the difference was not significant. (4) When normal cells were mixed with cells with reduced sialic acid content, the cells with less sialic acid settled significantly faster than those with more sialic acid. The present experiment may conclude that erythrocyte aggregation is induced preferentially among more deformable cells and/or among less negatively charged cells with weaker electrostatic repulsive force, and then the formed aggregates settle faster.

Gamma-ray-irradiated red blood cells stored in mannitol-adenine-phosphate medium: rheological evaluation and susceptibility to oxidative stress.

BACKGROUND AND OBJECTIVES: To evaluate the rheological properties and the oxidative susceptibility of gamma-ray-irradiated red blood cells (RBCs). MATERIALS AND METHODS: RBCs in mannitol-adenine-phosphate (MAP) medium were irradiated with 35 Gy and stored at 4 degrees C for 4 weeks. The deformability of the RBCs was examined under shear flow in relation to the morphological and biochemical changes. The RBCs were further exposed to 1 mM FeSO(4) and 5 mM ascorbate to examine the oxidative susceptibility. RESULTS: The RBC deformability was decreased during storage, and the impairment was further enhanced by the irradiation, which promoted cell shrinkage and intracellular hemoglobin condensation accompanying potassium loss. Lipid peroxidation and protein aggregation of the RBC membrane as well as echinocytosis were not enhanced by the irradiation. The exposure to free iron did not stimulate the oxidation of the irradiated RBC membrane. CONCLUSION: The decreased deformability of gamma-ray-irradiated RBCs in MAP medium was mainly induced by dehydration due to potassium loss, and the membrane lipids and proteins were stably preserved against oxidative stress.

Decreased deformability of the X-ray-irradiated red blood cells stored in mannitol-adenine-phosphate medium.

X-ray irradiation of blood is an effective way to prevent transfusion-associated graft-versus-host disease. Red blood cells (RBCs) from normal donors suspended in mannitol-adenine-phosphate (MAP) medium were irradiated with X-ray of 15 and 35 Gy in minimum dose. The change of deformability of the RBCs during storage at 4 degrees C for 4 weeks was examined under shear stress of 13-130 dyn/cm2 using a rheoscope, in relation to the hematological and biochemical properties. (1) The deformability of RBCs was decreased during the storage, and it was further decreased by the irradiation. In addition, the number of undeformable RBCs against a given shear stress increased after the irradiation. (2) The cell volume gradually decreased, while the intracellular hemoglobin concentration increased. These changes were accelerated by the irradiation. The echinocytic transformation during the storage was not accelerated by the irradiation. (3) The content of aggregated proteins reducible with beta-mercaptoethanol in RBC membrane increased during the storage, but was not increased by the irradiation. Membrane lipid peroxidation was not increased during the storage and by the irradiation. (4) Leakage of potassium ions from RBCs during the storage was accelerated by the irradiation. In conclusion, shear-induced deformation of RBCs stored in MAP medium was impaired by X-ray irradiation, mainly due to dehydration caused by excess leakage of potassium ions from RBCs.