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.

Elevated VEGF-plasma levels in young patients with mild essential hypertension.

BACKGROUND: Growing evidence shows that inflammation plays a pivotal role in the pathophysiology of essential hypertension (EH). Vascular endothelial cell growth factor (VEGF) is currently discussed as a possible mediator of inflammation. To investigate the hypothesis that VEGF plays a role as an inflammatory mediator in EH we performed the present pilot study of young patients in a very early stage of EH. MATERIALS AND METHODS: 15 young patients with mild EH [33.8 +/- 7.3 years, systolic blood pressure (SBP): 143.8 +/- 10.5 mmHg, diastolic blood pressure (DBP): 88.2 +/- 11.1 mmHg, mean arterial pressure (MAP) 106.6 +/- 10.4 mmHg] and 15 healthy controls (31.7 +/- 10.6 years) were examined. Blood was drawn from a peripheral vein and serum levels of VEGF, monocyte-chemoattractant-protein (MCP)-1, high-sensitivity C-reactive protein (hsCRP), interleukin (IL)-6, and tumour-necrosis-factor (TNF)-alpha were measured via commercially available enzyme-linked immunoassays. RESULTS: Hypertensives showed increased plasma levels of VEGF (P < 0.05) and MCP-1 (P < 0.05). VEGF positively correlated with MAP (r = 0.46, P < 0.05) and MCP-1 (r = 0.63, P < 0.01). Multivariate analysis demonstrated VEGF to be an independent predictor of MCP-1 levels. Furthermore, hypertensives had higher levels of hsCRP (P < 0.01), IL-6 (P < 0.001) and TNF-alpha (P < 0.05). IL-6 levels correlated with SBP (r = 0.59, P < 0.001), DBP (r = 0.67, P < 0.001) and MAP (r = 0.46, P < 0.001). A significant positive correlation was also found between hsCRP levels and SBP (r = 0.39, P < 0.05). CONCLUSIONS: This pilot study demonstrates that in an early state of EH, inflammatory pathways have already been activated. Besides classical pro-inflammatory cytokines, VEGF serum levels are significantly elevated. The positive correlation of VEGF with MCP-1 is suggestive for the already described induction of MCP-1 via VEGF.

Platelets contribute to enhanced MCP-1 levels in patients with chronic heart failure.

BACKGROUND: Increasing scientific data suggest a role for inflammation in chronic heart failure (CHF), but up to now the exact mechanisms are still not clear. Recently, platelets were identified as inducing inflammation partly by releasing cytokines. This new aspect necessitates further studies about the contribution of platelets for the inflammatory setting of CHF. METHODS: 50 CHF patients (mean 66.9 (SD 12.6) years, mean EF 22.1% (SD 9.1)) and 25 healthy controls (mean 63.6 (SD 10.2) years) were examined. MCP-1 serum levels were measured via EIA, expression of platelet CD154 by flow cytometry. In in-vitro experiments activated platelets were cocultured with human umbilical vein endothelial cells (HUVEC) in the presence and absence of anti-CD154 antibodies. MCP-1 in the supernatants was measured by EIA. RESULTS: CHF patients showed significantly enhanced MCP-1 levels (median: 191.8; 25th centile: 153.7; 75th centile: 227.1 pg/ml vs median: 101.0; 25th centile: 86.7; 75th centile: 117.5 pg/ml, p<0.001). MCP-1 levels positively correlated with severity of CHF. In the cell coculture model activated platelets were able to significantly induce MCP-1 release from HUVEC in a CD154-dependent manner. Furthermore, CHF patients showed enhanced platelet CD154 expression with a positive correlation with MCP-1 levels. Aspirin therapy had no influence on either CD154 expression or MCP-1 levels. CONCLUSIONS: Platelets can contribute to enhanced MCP-1 levels in CHF. MCP-1 is markedly elevated in serum of CHF patients showing a direct correlation with the severity of symptoms and the degree of left ventricular dysfunction. Further studies are required to test whether MCP-1 blocking or sophisticated anti-platelet strategies may represent new therapeutic options in CHF.

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.

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.

Upregulation of CD40-CD40 ligand (CD154) in patients with acute cerebral ischemia.

BACKGROUND AND PURPOSE: Inflammation and hypercoagulability contribute to the development of acute cerebral ischemia. Both can be mediated by the CD40 system. This study investigated whether the CD40 system and related mediators are upregulated in patients with transient ischemic attack (TIA) or stroke. METHODS: Seventeen patients with TIA, 60 patients with complete stroke, and 15 control subjects were investigated. CD154 and P-selectin were analyzed on platelets and CD40 on monocytes during and 3 months after acute cerebral ischemia by double-label flow cytometry. Blood concentrations of soluble CD154 and monocyte chemoattractant protein-1 (MCP-1) were evaluated. RESULTS: Our main findings are as follows: (1) patients with acute cerebral ischemia showed a significant increase of CD154 on platelets and CD40 on monocytes compared with controls; (2) plasma levels of soluble CD154 were significantly higher in these patients; (3) these patients had significantly higher numbers of prothrombotic platelet-monocyte aggregates; (4) the chemoattractant MCP-1 was significantly elevated in cerebral ischemia; and (5) at 3 months' follow-up, upregulation of CD154 still persisted in patients with previous acute cerebral ischemia. CONCLUSIONS: Patients with acute cerebral ischemia show upregulation of the CD40 system, which might contribute to the known proinflammatory, proatherogenic, and prothrombotic milieu found in these patients.

Patients with acute coronary syndromes express enhanced CD40 ligand/CD154 on platelets.

OBJECTIVE: To investigate whether CD40L/CD154 on platelets and soluble CD40L/CD154 may play a role in the inflammatory process of acute coronary syndromes. DESIGN AND SETTING: Observational study in a university hospital. PATIENTS: 15 patients with acute myocardial infarction, 25 patients with unstable angina, 15 patients with stable angina, and 12 controls. MAIN OUTCOME MEASURES: CD40L/CD154 on platelets, P-selectin/CD62P on platelets, soluble CD40L/CD154 serum concentrations. RESULTS: Mean (SD) CD40L/CD154 expression on platelets was 6.2 (2.8) MFI (mean fluorescence intensity) in the infarct group, 11 (3.3) MFI in the unstable angina group (p < 0.001 v infarction), 3.6 (0.9) MFI in the stable angina group (p < 0.01 v infarction; p < 0.001 v unstable angina), and 3.2 (1.0) MFI in the controls (p < 0.01 v infarction; p < 0.001 v unstable angina; NS v stable angina). Soluble CD40L/CD154 concentration was 5.2 (1.1) ng/ml in the infarct group, 4.2 (0.7) ng/ml in the unstable angina group (p < 0.001 v infarction), 2.9 (1.0) ng/ml in stable angina group (p < 0.001 v infarction and unstable angina), and 3.0 (0.5) ng/ml in the controls (p < 0.001 v infarction and unstable angina; NS v stable angina). At a six months follow up, there was lower expression of CD40L/CD154 on platelets in patients with unstable angina (12.3 (3.6) v 3.8 (1.2) MFI, p < 0.0001) and acute myocardial infarction (6.2 (2.8) v 3.5 (0.8) MFI, p < 0.01) compared with their admission values six months earlier. Patients with unstable angina who needed redo coronary angioplasty (PTCA) or who had recurrence of angina were characterised by increased CD40L/CD154 expression on platelets compared with the remainder of the study group (recurrence of angina: 12.7 (3.2) v 9.7 (1.6) MFI, p < 0.05; re-do PTCA: 14.3 (4.2) v 10.3 (2.1) MFI, p < 0.05). CONCLUSIONS: Both CD40L/CD154 on platelets and soluble CD40L/CD154 are raised in patients with unstable angina and myocardial infarction. These findings suggest that CD40-CD40L/CD154 interactions may play a pathogenic role in triggering and propagation of acute coronary syndromes.