Postprandial serum induces apoptosis in endothelial cells: Role of polymorphonuclear-derived myeloperoxidase and metalloproteinase-9 activity.
ABSTRACT Postprandial state is a pro-inflammatory condition associated with a transient impairment of endothelial function. Recent evidence suggests that myeloperoxidase (MPO) and matrix metalloproteinase-9 (MMP-9) are involved in the pathogenesis of inflammatory vascular diseases such as atherosclerosis. The present study was carried out to investigate whether a fat meal induces polymorphonuclear (PMN) activation and increases the plasma activity of MPO and MMP-9 and whether postprandial serum exerts pro-apoptotic effects on endothelial cells. Fifteen healthy young men underwent a high-fat challenge containing 60g butter. Blood samples were drawn before, and 1, 2, and 4h after the meal. Leukocyte reactive oxygen species (ROS) production, plasma MPO and MMP-9 activity, endothelial-derived soluble CD146 levels, and advanced oxidation protein product (AOPP) levels were determined. Human umbilical vein endothelial cells (HUVECs) were treated with human sera to evaluate mitochondrial membrane potential, ROS production, annexin PI staining, and caspase-3 activity. Triglycerides, ROS production, MPO activity, AOPP levels, pro-MMP-9 zymographic activity, and soluble CD146 levels significantly increased during the 4h after the test meal. Postprandial serum significantly decreased the mitochondrial membrane potential, and increased the rate of ROS production, the percentage of annexin-positive HUVECs, and caspase-3 activity. A strong relationship was observed between postprandial increase in PMN-derived MPO and pro-MMP-9 activity, and the increased rate of apoptosis of endothelial cells exposed to postprandial serum. Data show that postprandial serum exerts pro-apoptotic effects on endothelial cells. The close relationships between markers of endothelial cell apoptosis and MPO and pro-MMP-9 activity suggest that the latter may contribute to the development of fat meal induced endothelial damage.
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ABSTRACT: Reactive oxygen species (ROS) are produced as a by-product of cellular metabolic pathways and function as a critical second messenger in a variety of intracellular signaling pathways. Thus, a defect or deficiency in the anti-oxidant defense system on the one hand and/or the excessive intracellular generation of ROS on the other renders a cell oxidatively stressed. As a consequence, direct or indirect involvement of ROS in numerous diseases has been documented. In most of these cases, the deleterious effect of ROS is a function of activation of intracellular cell-death circuitry. To that end, involvement of ROS at different phases of the apoptotic pathway, such as induction of mitochondrial permeability transition and release of mitochondrial death amplification factors, activation of intracellular caspases and DNA damage, has been clearly established. For instance, the ROS-induced alteration of constitutive mitochondrial proteins, such as the voltage-dependent anion channel (VDAC) and/or the adenine nucleotide translocase (ANT) can induce the pro-apoptotic mitochondrial membrane permabilization. Not only do these observations provide insight into the intricate mechanisms underlying a variety of disease states, but they also present novel opportunities for the design and development of more effective therapeutic strategies.Histology and histopathology 02/2005; 20(1):205-19. · 2.28 Impact Factor
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ABSTRACT: We previously described the presence of advanced oxidation protein products (AOPP), a novel marker of oxidative stress in the plasma of hemodialyzed patients (HD). The present study was carried out to further investigate how myeloperoxidase (MPO)-catalyzed reactions could contribute to AOPP generation in the plasma. First, patterns of plasma protein oxidation obtained after in vitro incubation of control plasma with hypochlorous acid (HOCl) were compared to those from HD patients and control plasma. The use of various analytical techniques enabled localising and identifying the main oxidized proteins with albumin (HSA) after protein separation by size-exclusion chromatography and SDS-PAGE electrophoresis. The characterization of the oxidation level of the individual plasma proteins in terms of carbonyl groups and 3-nitrotyrosine formations was performed by immunoblotting. Secondly, to highlight the significance of AOPP index monitored by spectrophotometry, spectra were established for plasma fractions from HD patients and compared to data for control plasma and HOCl-treated plasma. The corresponding absorbance difference spectra were matched with external standards such as dityrosine, nitrotyrosine and pentosidine and elaborated chromophoric probe models. Indeed, HSA was chlorinated by HOCl reagent or HOCl generated via the MPO/H(2)O(2)/Cl(-) system and was nitrated by tetranitromethane. Increased absorbances at the range of 340 nm were observed both with chlorinated and nitrated HSA. Finally, our results indicate that HOCl, and not NO(2)(*), generated via MPO activity, could represent one of the pathways for AOPP production in plasma proteins exposed to activated phagocytes.Biochimica et Biophysica Acta 07/2004; 1689(2):91-102. · 4.66 Impact Factor
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ABSTRACT: 2',7'-Dichlorofluorescein and dihydrorhodamine 123 were evaluated as probes for detecting changes in intracellular H2O2 in cultured endothelial cells. Stable intracellular levels of these probes were established within 15 min of exposure to the probe in culture medium. With continued presence of the probe in the medium, intracellular levels were unchanged for 1 h. However, if medium without the probes was used after intracellular loading had occurred, there was a greater than 90% loss of intracellular dichlorofluorescin, dichlorofluorescein, and dihydrorhodamine 123 while intracellular rhodamine 123 decreased by only 15%. Exposure of endothelial cells to exogenous 100 microM H2O2 for 1 h increased intracellular rhodamine 123 by 83%, but there was a reproducible decrease of 53% in intracellular dichlorofluorescein. Exposure to 0.05 mM BCNU plus 10 mM aminotriazole for 2 h increased intracellular rhodamine 123 by 111%. In vitro studies of dihydrorhodamine 123 oxidation were similar to previous reports of dichlorofluorescin oxidation. Oxidation of dihydrorhodamine 123 does not occur with H2O2 alone, but is mediated by a variety of secondary H2O2-dependent intracellular reactions including H2O2-cytochrome c and H2O2-Fe2+. Our results suggest that detection of increased oxidation of these probes in endothelial cells is most useful as a marker of a change in general cellular oxidant production.Archives of Biochemistry and Biophysics 06/1993; 302(2):348-55. · 3.37 Impact Factor