Glucose-dependent leukocyte activation in patients with type 2 diabetes mellitus, familial combined hyperlipidemia and healthy controls.

BACKGROUND: Leukocyte activation has been associated with vascular complications in type 2 diabetes mellitus (T2DM). Hyperglycemia may be involved in this leukocyte activation. Our aim was to investigate the role of elevated glucose concentrations on leukocyte activation in patients with a wide range of insulin sensitivity. METHODS: Leukocyte activation was determined after ingestion of 75 gram glucose in subjects with T2DM, familial combined hyperlipidemia (FCH) and healthy controls. Leukocyte activation markers were measured by flow cytometry. Postprandial changes were calculated as the area under the curve (AUC), and the incremental area under the curve corrected for baseline values (dAUC). RESULTS: 51 Subjects (20 T2DM, 17 FCH and 14 controls) were included. Fasting neutrophil CD66b expression and CD66b-AUC were respectively 36% and 39% higher in T2DM patients than in controls (p=0.004 and p=0.003). Fasting neutrophil CD66b expression correlated positively with glucose-AUC (Spearman's rho 0.481, p<0.001) and HbA1c (rho 0.433, p=0.002). Although fasting monocyte CD11b expression was not significantly different between subjects, monocyte CD11b-AUC was 26% higher in T2DM than in controls (p=0.006). Similar trends were observed for FCH patients. Monocyte CD11b-dAUC correlated positively with glucose-AUC (rho 0.322, p=0.022) and HbA1c (rho 0.319, p=0.023). CONCLUSIONS: These data suggest that both acute and chronic hyperglycemia, associated with insulin resistance as seen in T2DM and FCH, are involved in the increased fasting and postprandial leukocyte activation observed in these conditions.

Differential complement activation pathways promote C3b deposition on native and acetylated LDL thereby inducing lipoprotein binding to the complement receptor 1.

Lipoproteins can induce complement activation resulting in opsonization and binding of these complexes to complement receptors. We investigated the binding of opsonized native LDL and acetylated LDL (acLDL) to the complement receptor 1 (CR1). Binding of complement factors C3b, IgM, C1q, mannose-binding lectin (MBL), and properdin to LDL and acLDL were investigated by ELISA. Subsequent binding of opsonized LDL and acLDL to CR1 on CR1-transfected Chinese Hamster Ovarian cells (CHO-CR1) was tested by flow cytometry. Both native LDL and acLDL induced complement activation with subsequent C3b opsonization upon incubation with normal human serum. Opsonized LDL and acLDL bound to CR1. Binding to CHO-CR1 was reduced by EDTA, whereas MgEGTA only reduced the binding of opsonized LDL, but not of acLDL suggesting involvement of the alternative pathway in the binding of acLDL to CR1. In vitro incubations showed that LDL bound C1q, whereas acLDL bound to C1q, IgM, and properdin. MBL did neither bind to LDL nor to acLDL. The relevance of these findings was demonstrated by the fact that ex vivo up-regulation of CR1 on leukocytes was accompanied by a concomitant increased binding of apolipoprotein B-containing lipoproteins to leukocytes without changes in LDL-receptor expression. In conclusion, CR1 is able to bind opsonized native LDL and acLDL. Binding of LDL to CR1 is mediated via the classical pathway, whereas binding of acLDL is mediated via both the classical and alternative pathways. Binding of lipoproteins to CR1 may be of clinical relevance due to the ubiquitous cellular distribution of CR1.

The postprandial situation as a pro-inflammatory condition / La situación posprandial como condición proinflamatoria

Postprandial lipemia has been associated with cardiovascular disease. The current pathophysiological concept is that postprandial remnant lipoproteins migrate into the subendothelial space and that remnants activate circulating leukocytes and endothelial cells. Activated monocytes adhere to endothelial adhesion molecules, facilitating subendothelial migration of monocytes. These cells differentiate into macrophages, with the risk of foam cell formation, due to uptake of remnants and modified lipoproteins. Evidence is emerging that specific interventions may reduce the atherogenic postprandial inflammation. Fruits rich in polyphenols, virgin olive oil, carotenoids and exercise have recently been found to reduce postprandial inflammation. Pharmaceutical interventions with fibrates or statins not only improve the overall lipid profile, but reduce postprandial inflammation as well. This review will deal with the current concept of postprandial inflammation in relation to the development of atherosclerosis and potential interventions to reduce postprandial inflammation

La lipidemia posprandial está relacionada con la enfermedad cardiovascular. El concepto patofisiológico actual es que las partículas remanentes traspasan el endotelio, activan los leucocitos y las células endoteliales. Los monocitos activados se adhieren a la paredendotelial por mediación de moléculas de adhesión, facilitando así la migración de los monocitos al espacio subendotelial. Estas células se transforman en macrófagos, convirtiéndose definitivamente en células espumosas después de haber internalizado las partículas remanentes y otras lipoproteínas modificadas. Recientes estudios sugieren que existen intervenciones efectivas para modular la inflamación posprandial, y de esta forma rebajar el riesgo cardiovascular. Frutas ricas en polifenoles, aceite de oliva virgen, el caroteno y el ejercicio son ejemplos que han demostrado una reducción de la inflamación posprandial. El tratamiento con estatinas y fibratos no solo mejora el perfil lipídico, sino que también rebaja la lipidemia posprandial. Esta revisión describe los recientes conceptos de la inflamación posprandial relacionada con la generación de ateroesclerosis y también trata las intervenciones que pueden influir positivamente en la inflamación posprandial

Postprandial inflammation: targeting glucose and lipids.

Many risk factors have been identified as being responsible for the process of atherogenesis. Several of these risk factors are related to inflammation, which is an obligatory feature of the atherosclerotic plaque. Increasing evidence suggests that postprandial lipoproteins and glucose may be involved in the inflammatory process preceding the development of atherosclerosis. During the postprandial situation, remnants of chylomicrons and very low-density lipoproteins bind to circulating leukocytes and endothelial cells, leading to a state of acute activation with the expression of integrins on different cells, the generation of oxidative stress, production of cytokines and complement activation. Elevated plasma glucose levels may also induce leukocyte activation in humans. In addition, advanced glycation end products, formed during hyperglycemia, cause inflammation and endothelial damage. This chain of events results in a situation of acute inflammation causing endothelial dysfunction, which may be one of the earliest defects in atherogenesis. Interestingly, while this may occur several times each day after each meal, there is only limited information on the contribution of different nutrients on the postprandial inflammatory processes. In this review, we will focus on the available evidence and we will discuss the role of lifestyle and pharmaceutical interventions in modulating postprandial inflammation.

The postprandial situation as a pro-inflammatory condition.

Postprandial lipemia has been associated with cardiovascular disease. The current pathophysiological concept is that postprandial remnant lipoproteins migrate into the subendothelial space and that remnants activate circulating leukocytes and endothelial cells. Activated monocytes adhere to endothelial adhesion molecules, facilitating subendothelial migration of monocytes. These cells differentiate into macrophages, with the risk of foam cell formation, due to uptake of remnants and modified lipoproteins. Evidence is emerging that specific interventions may reduce the atherogenic postprandial inflammation. Fruits rich in polyphenols, virgin olive oil, carotenoids and exercise have recently been found to reduce postprandial inflammation. Pharmaceutical interventions with fibrates or statins not only improve the overall lipid profile, but reduce postprandial inflammation as well. This review will deal with the current concept of postprandial inflammation in relation to the development of atherosclerosis and potential interventions to reduce postprandial inflammation.

Erythrocyte-bound apolipoprotein B in relation to atherosclerosis, serum lipids and ABO blood group.

INTRODUCTION: Erythrocytes carry apolipoprotein B on their membrane, but the determining factors of erythrocyte-bound apolipoprotein B (ery-apoB) are unknown. We aimed to explore the determinants of ery-apoB to gain more insight into potential mechanisms. METHODS: Subjects with and without CVD were included (N = 398). Ery-apoB was measured on fresh whole blood samples using flow cytometry. Subjects with ery-apoB levels ≤ 0.20 a.u. were considered deficient. Carotid intima media thickness (CIMT) was determined as a measure of (subclinical) atherosclerosis. RESULTS: Mean ery-apoB value was 23.2% lower in subjects with increased CIMT (0.80 ± 0.09 mm, N = 140) compared to subjects with a normal CIMT (0.57 ± 0.08 mm, N = 258) (P = 0.007, adjusted P<0.001). CIMT and ery-apoB were inversely correlated (Spearman's r: -0.116, P = 0.021). A total of 55 subjects (13.6%) were considered ery-apoB deficient, which was associated with a medical history of CVD (OR: 1.86, 95% CI 1.04-3.33; adjusted OR: 1.55; 95% CI 0.85-2.82). Discontinuation of statins in 54 subjects did not influence ery-apoB values despite a 58.4% increase in serum apolipoprotein B. Subjects with blood group O had significantly higher ery-apoB values (1.56 ± 0.94 a.u.) when compared to subjects with blood group A (0.89 ± 1.15 a.u), blood group B (0.73 ± 0.1.12 a.u.) or blood group AB (0.69 ± 0.69 a.u.) (P-ANOVA = 0.002). CONCLUSION: Absence or very low values of ery-apoB are associated with clinical and subclinical atherosclerosis. While serum apolipoprotein B is not associated with ery-apoB, the ABO blood group seems to be a significant determinant.

Trans-vessel gradient of myeloperoxidase in coronary artery disease.

BACKGROUND: Coronary artery disease (CAD) may reflect generalized inflammation. We evaluated leucocyte activation in subjects with and without CAD in different vascular compartments. MATERIALS AND METHODS: Patients were divided in two groups; subjects without CAD (controls; n = 25) and with stable CAD (n = 52) based on coronary angiography. After blood sampling from vessels, cardiovascular risk factors and leucocyte activation markers CD11b, CD66b and cytoplasmatic myeloperoxidase (MPO) were determined by flow cytometry. RESULTS: Myeloperoxidase (MPO) was higher in patients with CAD at all sites compared with controls (188 ± 7 vs. 210 ± 12 au for venous (P < 0.05), 178 ± 7 vs. 212 ± 12 au for femoral artery (P = 0.08), 166 ± 7 vs. 195 ± 12 au for abdominal artery (P < 0.05), 166 ± 6 vs. 189 ± 14 au for left coronary (P = 0.08) and 163 ± 6 vs. 193 ± 12 au for the right coronary artery (P < 0.05)). Other markers did not differ between the groups. A gradient of inflammation from peripheral vessels to the coronaries was found by differences in MPO in both groups; from 210 ± 12 au in the venous compartments towards 189 ± 14 and 193 ± 12 au, in the left and right coronaries, respectively, for the controls (P = 0.001), and from 188 ± 7 au in the venous compartment towards 166 ± 6 and 163 ± 6 au in the left and right coronaries, respectively, for the patients (P = 0.007). Other leucocyte activation markers did not show such a gradient. CONCLUSIONS: There is a generalized inflammatory neutrophil gradient for MPO from peripheral vessels towards the coronaries in both patients with CAD and controls. However, patients with CAD show a higher degree of inflammation, mostly in the coronaries. These data strengthen the role of activated neutrophils in CAD.

Exploring the value of apoB48 as a marker for atherosclerosis in clinical practice.

BACKGROUND: Postprandial accumulation of atherogenic remnants has been described in patients with type 2 diabetes mellitus (T2DM), familial combined hyperlipidaemia (FCH), familial hypercholesterolaemia (FH) and coronary artery disease (CAD). Scarce data are available on fasting plasma apolipoprotein (apo) B48 levels in relation to these conditions and atherosclerosis. DESIGN: Treated patients with FCH (18), FH (20), T2DM (26), CAD (65), T2DM with CAD (T2DM/CAD) (28) and 33 healthy controls were included. Intima-media thickness (IMT) measurements were carried out to investigate subclinical atherosclerosis. RESULTS: LDL-C and total apoB were lowest in patients with T2DM/CAD owing to the more frequent use of lipid-lowering medication. Fasting plasma apoB48 was elevated in patients with FCH (11·38 ± 1·50 mg/L) and T2DM/CAD (9·65 ± 1·14 mg/L) compared with the other groups (anova, P < 0·01). CAD patients (8·09 ± 0·57 mg/L) had higher apoB48 levels than controls (5·74 ± 0·55 mg/L) and FH patients (5·40 ± 0·51 mg/L) (P = 0·02). IMT was highest in subjects with T2DM/CAD (0·77 ± 0·03 mm) (P < 0·01). The lowest IMT was measured in controls (0·56 ± 0·02 mm) and FCH patients (0·60 ± 0·03 mm). In the total group, the best association for apoB48 was found with fasting triglyceride (Pearson's r = 0·72, P < 0·001). In the subjects not using statins (n = 74), the best correlation was found with IMT (r = 0·52; P < 0·001), whereas total apoB was not associated with IMT (r = 0·20, P = 0·12). CONCLUSIONS: ApoB48 concentrations are highest in patients with FCH and in atherosclerotic subjects with T2DM. In patients not using statins, the surrogate atherosclerosis marker IMT correlates best with apoB48, suggesting that fasting apoB48 may help to detect subjects at risk.

Erythrocyte-associated apolipoprotein B and its relationship with clinical and subclinical atherosclerosis.

BACKGROUND: Apolipoprotein (apo) B-containing lipoproteins are closely linked to atherogenesis. These lipoproteins are transported in plasma and are also associated with blood leucocytes. Our aim was to investigate whether apoB-containing lipoproteins are also present on the surface of erythrocytes and investigate the relationship with the presence of atherosclerosis in a cross-sectional study. MATERIALS AND METHODS: Erythrocyte-bound apoB (ery-apoB) was measured by flowcytometry in subjects with (CAD+) and without coronary artery disease (CAD-), based on coronary angiography or on a history of cardiovascular disease. Intima media thickness (IMT) measurements were carried out using B-mode ultrasound. The relationship between ery-apoB and clinical and subclinical atherosclerosis was evaluated with binary logistic regression. RESULTS: A total of 166 subjects were included (40 CAD+ and 126 CAD-). ApoB was detected on freshly isolated erythrocytes (range: 0·1-5·5 au; mean ± SEM 0·86 ± 0·09 au) in all but nine subjects (four CAD+ and five CAD-). Ery-apoB was lower in CAD+ (0·62 ± 0·09 au) compared to CAD- (1·18 ± 0·10 au; P < 0·001). Higher ery-apoB was associated with a lower risk of CAD (adjusted OR: 0·003 (95% CI: 0·001-0·08; P < 0·001), but the protective effect was diminished with increasing age (adjusted OR: 1·10 (95% CI: 1·04-1·16; P < 0·001). IMT was increased in CAD+ subjects (0·77 ± 0·13 mm) compared to CAD- (0·57 ± 0·14 mm; P < 0·001). A significant negative association was found between ery-apoB and IMT (ß = -0·214: 95% CI -0·284 to -0·145; P < 0·001). There was no association between ery-apoB and plasma apoB (Pearson's r = -0·45; P = 0·57). CONCLUSIONS: Human erythrocytes carry apoB-containing lipoproteins. Subjects with atherosclerosis have lower ery-apoB. High ery-apoB may be protective against atherosclerosis and may reflect an alternative blood cell-mediated lipoprotein transport system in the circulation, in which these lipoproteins less likely interact with the endothelium.

NOX2, p22phox and p47phox are targeted to the nuclear pore complex in ischemic cardiomyocytes colocalizing with local reactive oxygen species.

BACKGROUND: NADPH oxidases play an essential role in reactive oxygen species (ROS)-based signaling in the heart. Previously, we have demonstrated that (peri)nuclear expression of the catalytic NADPH oxidase subunit NOX2 in stressed cardiomyocytes, e.g. under ischemia or high concentrations of homocysteine, is an important step in the induction of apoptosis in these cells. Here this ischemia-induced nuclear targeting and activation of NOX2 was specified in cardiomyocytes. METHODS: The effect of ischemia, mimicked by metabolic inhibition, on nuclear localization of NOX2 and the NADPH oxidase subunits p22(phox) and p47(phox), was analyzed in rat neonatal cardiomyoblasts (H9c2 cells) using Western blot, immuno-electron microscopy and digital-imaging microscopy. RESULTS: NOX2 expression significantly increased in nuclear fractions of ischemic H9c2 cells. In addition, in these cells NOX2 was found to colocalize in the nuclear envelope with nuclear pore complexes, p22(phox), p47(phox) and nitrotyrosine residues, a marker for the generation of ROS. Inhibition of NADPH oxidase activity, with apocynin and DPI, significantly reduced (peri)nuclear expression of nitrotyrosine. CONCLUSION: We for the first time show that NOX2, p22(phox) and p47(phox) are targeted to and produce ROS at the nuclear pore complex in ischemic cardiomyocytes.