Influence of High Polyphenol Beverage on Stress-Induced Platelet Activation.

OBJECTIVES: Platelets are playing a crucial role in acute cardiovascular events. We investigated if physical stress activates platelets and whether this activation can be inhibited by a polyphenol-enriched diet. METHODS: Blood samples were taken from a total of 103 athletes three weeks before, one day before, immediately as well as 24 hours and 72 hours after a marathon run. Participants were randomized, double-blinded and divided into two groups. One group received a polyphenol-rich beverage the other the same beverage without polyphenols. Besides analysis of platelet counts and impedance-aggregometric-measurement of platelet activity, soluble P-selectin and Endothelin-A measurements were performed. RESULTS: In the control group, runners showed a 2.2-fold increased platelet aggregation directly after completing a marathon and within the following three days when compared with baseline values (p<0.01). In accordance, significant increases in sP-selectin (57.52ng/ml vs. 94.86ng/ml;p<0.01) were detectable. In contrast, for the group consuming a beverage with increased polyphenol content (upper quartile of study beverage intake) we did not find any increase of platelet aggregation. DISCUSSION: Physical stress causes a significant increase in platelet activity. Our results demonstrate that a diet enriched in polyphenols is capable of preventing platelet activation. These findings might indicate a diminished cardiovascular stress-reaction following pre-exposition to polyphenol-enriched diet.

Tissue engineering of endothelial cells and the immune response.

BACKGROUND: While tissue engineering offers promise for organ and tissue transplantation, it can also be used to examine transplant and immune biology. Endothelial cells engrafted within 3-dimensional matrices create stable units that produce all of the factors of a functional quiescent endothelium. Perivascular implantation of tissue engineered endothelial cell constructs provides long-term control of vascular repair after injury. This control is established without restoration of the natural luminal:mural endothelium, and most intriguingly, without engendering host allo- and xenogeneic immune responses. We examined how endothelial immunogenicity is controlled by interaction with 3-dimensional matrices. MATERIALS AND METHODS: Human aortic endothelial cells (HAE) were either grown to confluence on polystyrene tissue culture plates or within 3-dimensional collagen-based matrices. Major histocompatibility complex (MHC) class II, integrin, interferon (IFN)-gamma receptor expression, and signaling were analyzed via confocal microscopy, flow cytometry, reverse transcription polymerase chain reaction (RT-PCR), and microarray. Splenocyte proliferation was assayed by thymidine incorporation. RESULTS: Despite similar expression levels of IFN-gamma receptors, matrix-embedded HAE elicited far less STAT-1 phosphorylation upon IFN-gamma stimulation, and expressed 2-fold less MHC II than HAE grown to confluence on culture plates (P < .001). This effect correlated with reduced expression of integrin alpha(v) and beta(3) (P < .002), and muted proliferation of porcine splenocytes (P < .001). CONCLUSIONS: Matrix architecture is critical for modulation of endothelial immunogenicity. Embedding HAE within a physiologic 3-dimensional environment affects activity of intracellular signaling pathways, MHC II expression, and subsequent activation of immune cells. These findings might offer novel insights into our understanding of endothelial-mediated diseases and might enhance our ability to leverage the potential for cell-based therapies.

Acute effect of H.E.L.P. treatment on radical scavenging enzyme activities, total glutathione concentrations in granulocytes, and selenium in plasma.

BACKGROUND: It has been suggested that granulocytes are activated on artificial surfaces such as dialyzer membranes or by plasma separation procedures resulting in the generation of free radicals. We reported recently that free radical scavenging enzyme (FRSE) activities of red blood cells obtained from patients undergoing hemodialysis and LDL-apheresis (LA) do not reflect an acute oxidative stress. However, because mature red cells are free of DNA and RNA, enzymes cannot be regulated on the gene level. In contrast, granulocytes are nucleated cells in which genes can be regulated, e. g. by redox sensitive transcription factors activated by extracellular oxidative stress. Therefore, granulocyte FRSE may better reflect acute oxidative stress caused by extracorporeal treatment. MATERIALS AND METHODS: Hyperlipidemic patients (n = 18) with coronary heart disease (CHD) were treated with the Heparin-induced-Extracorporeal-LDL-Precipitation (H.E.L.P.) system. Glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), superoxide dismutase (SOD) activities, and total glutathione were determined in granulocytes before and immediately after a single LA treatment. Selenium (Se) concentrations were assessed in plasma. RESULTS: As a result of the H.E.L.P. treatment GSSG-R activity was significantly induced (+ 20%) and the GSH concentration increased (+ 41%) in granulocytes. GSH-Px activity in granulocytes (- 19%) and Se in plasma (- 27%) were significantly reduced whereas SOD activity in granulocytes was not affected by the H.E.L.P. procedure. CONCLUSION: These results show that the defence against oxygen radicals in granulocytes is affected but not severely compromised in patients undergoing regular H.E.L.P-LDL-apheresis treatment, which points to the safety of this system with respect to oxidative stress.

Review: the oxidant/antioxidant balance during regular low density lipoprotein apheresis.

Low density lipoprotein (LDL) apheresis is a safe procedure to treat severe hypercholesterolemia in patients with chronic heart disease (CHD). However, both hypercholesterolemia and extracorporeal treatment have been associated with oxidative stress. Even though LDL lowering has been proven to reduce CHD, the oxidative modification of LDL has been suggested to render these lipoproteins more atherogenic. It is therefore important to know whether LDL apheresis is safe with respect to oxidative stress including LDL oxidation. The contact of living cells such as leukocytes with artificial surfaces during extracorporeal treatment induces the liberation of various chemokines and cytokines as well as oxygen-derived radicals also known as respiratory burst. These effects justify the consideration of leukocyte activation resulting from extracorporeal treatment as an inflammatory reaction. In extracorporeal circuits such as those used for hemodialysis, the release of oxygen radicals has been shown and depends on the fiber material used in the dialyzer membranes. Reactive oxygen radicals can interact with different cell components such as carbohydrates, DNA, proteins, and lipids. Antioxidants in the form of low molecular weight molecules such as glutathione or radical scavenging enzymes such as superoxide dismutase offer protection against the damaging effects of prooxidants. The disturbed balance between prooxidants and antioxidants is considered as oxidative stress. Therefore, either an increase in oxygen radical formation or a decrease of antioxidants will lead to oxidative stress. During LDL apheresis, a decrease of low molecular weight antioxidants has been reported. In contrast, we have observed an increase in plasma glutathione concentrations but no severe reduction in the activity of antioxidant enzymes in plasma, red cells, or granulocytes, which may explain the lack of plasma lipid peroxidation shown during this kind of extracorporeal treatment. In addition, LDL isolated at the end of apheresis procedures are more resistant to oxidation. These findings suggest that LDL apheresis is safe with respect to radical mediated injury.

Oxidative stress during dialysis: effect on free radical scavenging enzyme (FRSE) activities and glutathione (GSH) concentration in granulocytes.

BACKGROUND: Living cells are protected by free radical scavenging enzymes against oxygen radical-mediated damage. It has been suggested that granulocytes are activated on the surface of dialyser membranes, resulting in the generation of free radicals. We have recently reported a lack of plasma lipid peroxidation and unchanged glutathione peroxidase (GSH-Px) as well as glutathione reductase (GSSG-R) activities in red blood cells of haemodialysis patients. However, because mature red cells are free of DNA and RNA, free radical scavenging enzymes (FRSE) cannot be regulated on the gene level in response to an acute oxidative stress. In contrast to erythrocytes, granulocytes are nucleated cells and FRSE protein concentrations can therefore be modulated. METHODS: GSH-Px, GSSG-R, superoxide dismutase (SOD) activities and total glutathione (GSH) were determined spectrophotometrically using a Cobas Fara semi-automatic analyser in granulocytes of 31 healthy blood donors and in 28 patients with chronic renal failure (CRF) for more than 6 months before as well as immediately after a single dialysis treatment. Patients were treated either by haemodialysis (n = 17) using low-flux polysulphone membranes or by haemofiltration (n= 1l) usings high-flux polysulphone membranes. RESULTS: Compared to healthy controls, SOD and GSSG-R activities were increased in granulocytes of HD and HF patients, GSH and GSH-Px were decreased before a single treatment. After dialysis SOD and GSH-PX activities were significantly induced by both HD and HF whereas GSSG-R activities and GSH were decreased. CONCLUSIONS: These results show that the enzymatic defence against oxygen radicals can be induced in granulocytes of patients undergoing regular dialysis treatment, whereas the non-enzymatic defence is compromised as shown by decreased GSH concentrations, both suggesting increased oxidative stress.

Activity of free radical scavenging enzymes in red cells and plasma of patients undergoing extracorporeal low-density lipoprotein apheresis.

There is evidence that reactive oxygen species (ROSs) are generated in extracorporeal circuits. Free radical scavenging enzymes (FRSEs) such as glutathione reductase (GSSG-R), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) protect against the damaging effect of ROSs. The influence of extracorporeal treatment on FRSE activity was investigated in the plasma and red blood cells (RBCs) of 21 patients undergoing regular low-density lipoprotein (LDL) apheresis. The FRSEs GSSG-R, GSH-Px, and SOD were measured. Determinations were made before and after a single treatment. Because all apheresis patients suffered from coronary heart disease (CHD), 201 CHD patients and 90 individuals without CHD, neither group undergoing apheresis, served as controls. In apheresis patients, GSH-Px (33.9+/-8.2 U/g Hb) and GSSG-R (7.6+/-0.9 U/g Hb) activities were increased whereas SOD activity (5.4+/-1.5 U/g Hb) was decreased in RBCs before a single treatment compared to controls. Plasma FRSEs of apheresis patients were not different from those of controls. There was no effect of a single treatment on FRSEs in RBCs. However, a significant decrease in plasma GSH-Px activity (209.9+/-24.9 U/ml) due to the extracorporeal treatment was observed. These data show that long-term extracorporeal therapy with LDL apheresis modulates the activity of antioxidant enzymes in RBCs whereas a single treatment was without major effect on FRSE activity in RBCs and plasma, except for plasma GSH-Px.