Thorsten Kälsch

Endotoxin-induced effects on platelets and monocytes in an in vivo model of inflammation

AimsChronic inflammation is a major contributing factor to atherosclerosis and various markers of inflammation, fibrinolysis and coagulation are upregulated in patients with established atherosclerotic disease. The aim of this study was to investigate the direct and short-term effects of inflammation on platelet and monocyte activation with an in vivo model of endotoxemia in healthy volunteers.Methods and resultsIn this study, 13 healthy male subjects with a mean age of 29.5±5.4 years received intravenous administration of lipopolysaccharide (LPS; 20 IU/kg IV). The kinetics of CD40-ligand and CD62P expression on platelets, tissue-factor binding on monocytes and platelet-monocyte aggregates were measured by whole blood flow cytometry at baseline and at 1, 2, 4, 6 and 24 hours after LPS administration. Plasma levels of soluble CD40-ligand were measured with an ELISA over the same time course. Platelet-monocyte aggregates, tissue-factor binding on monocytes and surface expression of platelet CD40L significantly increased in experimental endotoxemia in vivo, reaching peak values 1 hour after LPS administation. All values returned to baseline after 24 hours. Surface expression of CD62P on platelets and plasma levels of sCD40L did not change significantly in response to LPS.ConclusionsIn vivo administration of endotoxin leads to an activation of platelets and monocytes with an upregulation of proatherogenic CD40L on platelets. These findings underpin the role of inflammation in early atherogenesis through platelet and monocyte activation in an in vivo model.

Impact of fibrinogen concentration in severely ill patients on mechanical properties of whole blood clots.

Fibrinogen concentration influences mechanical and functional properties of the clot. The purpose of the present study was to identify threshold concentrations of fibrinogen resulting in relevant changes in whole blood clot elastic modulus and platelet contractile force, as well as plasma prothrombin time and activated partial thromboplastin time. We measured clot elastic modulus, platelet contractile force, and other hemostasis parameters in whole blood samples from 552 patients admitted to a surgical intensive care unit. Platelet contractile force and clot elastic modulus were measured using the Hemodyne apparatus. Fibrinogen levels were between less than 0.10 and 9.44 g/l, with a mean of 2.41 g/l. Mean platelet count was 203 × 109 l−1, with a range of 16 × 109 l−1 to 682 × 109 l−1. High levels of fibrinogen result in improved mechanical stability and improved interaction of platelets with the fibrin network. Clot elastic modulus and platelet contractile force are correlated positively with plasma fibrinogen concentration. However, there was no threshold concentration or ceiling effect concerning the mechanical properties of the clots. In contrast, clotting time assays such as prothrombin time, thrombin time, or activated partial thromboplastin time are influenced by the fibrinogen concentration only at levels below 1 g/l. In linear regression analysis, clot elastic modulus was mainly influenced by fibrinogen concentration (F = 185.4, P < 0.0001), whereas platelet contractile force was influenced by fibrinogen (F = 197.0, P < 0.0001) and platelet count (F = 104.7, P < 0.0001). The present data show that 1 g/l is a threshold fibrinogen concentration for an effect on coagulation assays such as prothrombin time, thrombin time, or activated partial thromboplastin time, but increasing fibrinogen concentrations above this level results in further continuous improvement of mechanical properties of the whole blood clot.

High plasma levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and interleukin-8 (IL-8) characterize patients prone to ventricular fibrillation complicating myocardial infarction.

Abstract Background: Atherosclerotic plaques prone to cause thrombotic complications and plaque rupture account for the majority of fatal myocardial infarctions (MI), which may be complicated by ventricular fibrillation (VF). Matrix-degrading metalloproteinases (MMPs) and their inhibitors (TIMPs) are expressed in atherosclerotic lesions and contribute to plaque vulnerability. Interleukin-8 (IL-8) is one of the predominant chemokines interacting with MMPs and TIMPs and the coagulation system. The aim of the present study was to assess potential differences of levels of MMP-9, TIMP-1 and IL-8 in postmyocardial infarction patients with or without VF complicating acute MI. Methods: Blood samples were taken from 45 patients with VF complicating acute MI and from 88 patients without VF. All samples were collected during a symptom-free interval remote from the acute ischemic event with a median of 556 days. The markers of interest were TIMP-1, MMP-9 and IL-8. Results: IL-8 and TIMP-1 levels were significantly higher among patients with VF than among patients without VF (p<0.001). In a logistic regression approach IL-8 was an independent indicator of patients prone to VF during MI (p=0.03). High levels of TIMP-1 (p=0.05), MMP-9 (p=0.03), the MMP-9/TIMP-1 ratio (p=0.049) and hypertension (p=0.02) were found to be indicators in patients with reinfarction or unstable angina pectoris during follow-up. Hypertension (p=0.02) and MMP-9 (p=0.03) were the only significant indicators characterizing patients undergoing coronary reinterventions, such as percutaneous coronary interventions and coronary bypass surgery. Conclusions: Higher TIMP-1 and IL-8 levels are present in patients with VF complicating MI. High TIMP-levels may be related to the degree of fibrosis which is a substrate for electrical instability and may contribute to the occurrence of VF. Patients prone to develop VF during MI seem to have an increased proinflammatory condition compared to patients without VF. Clin Chem Lab Med 2007;45:1360–5.

1α,25-Dihydroxyvitamin D3 Attenuates Platelet Activation and the Expression of VCAM-1 and MT1-MMP in Human Endothelial Cells

Objective: Inflammatory conditions contribute to increased expression of various activity markers in platelets and endothelial cells, leading to atherosclerotic changes in the vascular wall. The objective of this study was to investigate possible protective effects of 1α,25-dihydroxyvitamin D3 in an endothelial cell model. Methods: After a 24-hour incubation with 1α,25-dihydroxyvitamin D3, human umbilical vein endothelial cells were stimulated with lipopolysaccharide (LPS) and incubated in direct contact with platelets. The expression of CD40L and CD62P in platelets, the expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1 (VCAM-1), the urokinase receptor uPAR and membrane type 1 matrix metalloproteinase (MT1-MMP) in endothelial cells and endothelial cell reactive oxygen species generation were measured by flow cytometry. Endothelial nitric oxide synthase was analyzed by Western blot. Results: The increased expression of VCAM-1 and MT1-MMP in endothelial cells by proinflammatory stimulation with LPS and by direct contact with activated platelets was significantly reduced through preincubation with 1α,25-dihydroxyvitamin D3. Platelets in direct contact with preincubated endothelial cells showed significantly reduced CD62P expression when compared to platelets incubated with untreated endothelial cells. Conclusions: 1α,25-Dihydroxyvitamin D3 attenuates platelet activation and the expression of VCAM-1 and MT1-MMP in human endothelial cells and could have early therapeutic relevance in atherosclerotic diseases.

Simvastatin and atorvastatin attenuate VCAM-1 and uPAR expression on human endothelial cells and platelet surface expression of CD40 ligand.

BACKGROUND In addition to their cholesterol lowering ability, statins have proven pleiotropic effects in the cardiovascular system. Chronic inflammation with interactions between platelets and endothelial cells leads to an upregulation of activity markers of atherosclerosis. The purpose of this study was to investigate the effects of simvastatin and atorvastatin on platelets and endothelial cells in an in vitro endothelial cell model. METHODS AND RESULTS After a 24 h incubation period with either simvastatin or atorvastatin (1 μmol/L), human umbilical vein endothelial cells were stimulated for 1 h with lipopolysaccharide (LPS), and were then incubated in direct contact with activated platelets. Platelet surface expression of CD40L and CD62P and expression of ICAM-1, VCAM-1, uPAR and MT1-MMP on endothelial cells were measured by flow cytometry. Supernatants were analyzed by ELISA for soluble MMP-1. The increased expression of VCAM-1 and uPAR on endothelial cells by stimulation with LPS and by direct contact with activated platelets was significantly reduced to a similar extent through pre-incubation with both atorvastatin and simvastatin (p < 0.05). Platelets without endothelial cell contact, but in direct contact with either statin, showed similar significant reductions in surface expression of CD40L (p < 0.005). CONCLUSIONS These effects may explain the ability of statins to reduce the progression of atherosclerosis in addition to their cholesterol-lowering properties.

Use of Highly Sensitive C-Reactive Protein for Followup of Wegener’s Granulomatosis

Objective. Since Wegener’s granulomatosis (WG) represents a relapsing disease, efforts have been made to reliably predict relapses using blood tests. Followup measures such as conventionally determined C-reactive protein (CRP), antineutrophil cytoplasmic antibody (C-ANCA) titer, and proteinase-3 (PR3) ELISA are applied. We evaluated whether during remission elevated highly sensitive CRP (hsCRP) precedes relapse as a marker of subclinical inflammation and thus might improve clinical assessment. Methods. We investigated 227 sera of 57 patients with WG: 74 sera collected from patients in remission who subsequently relapsed (before relapse), 30 sera collected during relapse, and 123 sera from patients in remission without relapse. We also distinguished between major and minor relapse. hsCRP, conventionally determined CRP (CRP), C-ANCA, PR3-ELISA, and erythrocyte sedimentation rate (ESR) were measured using commercial kits, and levels were correlated to clinical status. Results. Only hsCRP and ANCA titer, but not CRP levels, were higher in sera from patients who subsequently relapsed versus those who did not, indicating patients at risk. Levels of hsCRP, CRP, and ESR were higher in sera collected during relapse than in the sera before relapse. hsCRP, conventional CRP, and ESR were also higher in samples collected during major relapse than before major relapse. Looking at the levels just before relapse compared to previous levels during remission, none of these measures rose directly before the clinical manifestation of the relapse. Conclusion. Our study provides evidence for an additional value of hsCRP in the clinical assessment of patients with WG.

Enhanced proinflammatory response of mononuclear cells to in vitro LPS-challenge in patients with ventricular fibrillation in the setting of acute myocardial infarction

AIMS Ventricular fibrillation (VF) in the setting of acute myocardial infarction (AMI) is the leading cause of sudden cardiac death. A potential role of intrinsic, subclinical inflammatory states in patients suffering from ischemia-related VF has not been investigated yet. The aim of the present study was (i) to examine serum levels of proinflammatory markers in VF survivors and (ii) to evaluate basal and lipopolysaccharide (LPS)-stimulated interleukin-8-mRNA (IL-8-mRNA) levels in patients with and without VF complicating AMI. METHODS Twenty-five patients with a history of VF during AMI and a control group of 25 AMI patients without VF were included. Blood samples were taken remote from AMI with a mean of 590 days. Circulating serum levels of IL-8, IL-6, soluble E-selectin (sE-selectin), tissue factor activity (TFA), tissue inhibitor of matrix-metalloproteinase-1 (TIMP-1) and matrix-metalloproteinase-9 (MMP-9) were measured. Mononuclear cells were isolated by density gradient centrifugation. The cells were stimulated with lipopolysaccharide (LPS) from Escherichia coli (700 ng/mL). IL-8-mRNA levels in mononuclear cells were determined by a colorimetric mRNA quantification assay. RESULTS Serum levels (median; range) of IL-8 (VF: 2.24 pg/mL; <0.10-19.3 pg/mL versus controls: 0.10 pg/mL; <0.10-7.7 pg/mL; p=0.014), IL-6 (VF: 0.68 pg/mL; <0.05-2.9 pg/mL versus controls: 0.23 pg/mL; <0.05-1.8 pg/mL; p=0.042) and TIMP-1 (VF: 229 ng/mL; 144-348 ng/mL versus controls: 186 ng/mL; 126-263 ng/mL; p=0.014) were significantly higher among patients with VF as compared to controls. Baseline IL-8-mRNA concentrations of blood mononuclear cells were significantly higher among patients with VF (257 amol/mL; 52-2672 amol/mL) as compared to patients without VF (37 amol/mL, 3.2-770 amol/mL; p<0.01). IL-8-mRNA levels after LPS-challenge were significantly higher among patients with VF (3503 amol/mL; 215-13,573 amol/mL) than in patients without VF (1003 amol/mL; 208-3386 amol/mL; p<0.01). CONCLUSIONS Circulating IL-8, IL-6, and TIMP-1 concentrations as well as IL-8-mRNA expression in mononuclear cells at baseline and after LPS-challenge are increased among patients with a history of VF in the setting of AMI as compared to patients without VF. These findings indicate an enhanced inflammatory response to a proinflammatory stimulus in VF survivors. The magnitude of this increased acute phase reactants may indicate a novel pathway of arrhythmogenesis in patients with AMI.

Elevation of the glycoxidation product Nε-(carboxymethyl)lysine in patients presenting with acute myocardial infarction

Abstract Background: An important role in the acceleration of vascular disease has been previously suggested for advanced glycation end products. Nε-(carboxymethyl)lysine (CML) is an advanced glycation end product formed on protein by combined non-enzymatic glycation and glycoxidation reactions. CML reacts with the receptor of advanced glycation end products inducing impairment of endothelium dependent relaxation and is a marker of oxidative stress. Methods: A total of 40 patients with acute myocardial infarction (17 patients with ST-elevation myocardial infarction, 23 patients with non-ST-elevation myocardial infarction) and 40 patients with stable coronary artery disease were included consecutively in this study. During coronary angiography, peripheral venous blood sample was taken for measuring CML. Results: Serum levels of CML were significantly increased in patients with acute myocardial infarction [17.9±10.7 vs. 6.6±3.1 arbitrary units (AU)/mg protein, p<0.001]. A cut-off value of CML>9.5 AU/mg protein was associated with an odds ratio of acute myocardial infarction of 39.7 [95% confidence interval (CI): 11.1–142, p<0.001], a sensitivity of 0.85 (95% CI: 0.70–0.94) and a specificity of 0.88 (95% CI: 0.73–0.96). Conclusions: CML levels are significantly elevated in patients presenting with acute myocardial infarction. These results suggest the involvement of endothelial dysfunction (through receptor interaction) and oxidative stress in acute myocardial infarction. Clin Chem Lab Med 2009;47:446–51.

Sudden death: Do cytokines and prothrombotic peptides contribute to the occurrence of ventricular fibrillation during acute myocardial infarction?

AIMS Sudden cardiac death (SCD) is frequently caused by ventricular fibrillation (VF) occurring in the course of acute myocardial infarction (AMI). It has not been investigated yet, to what extent markers of coagulation activation and inflammation differ between patients with and without VF in the acute phase of AMI.

Platelet and monocyte activation in acute ischemic stroke--is there a correlation with stroke etiology?

An upregulation of platelet CD40 ligand (CD40L) and CD62P has been described in atherosclerotic cardiovascular diseases and among patients with acute cerebral ischemia. Correlation between platelet and monocyte activation and the etiology of ischemic stroke were examined in 41 patients with acute ischemic stroke. Compared to 10 controls, all patients with stroke showed a significantly elevated platelet expression of CD40L (P < .001) and had significantly higher amounts of platelet–monocyte aggregates (P = .002). Plasma levels of interleukin 7 were significantly lower in patients with stroke compared to controls (P = .006). Patients with small artery disease had a significantly higher platelet CD40L expression than patients with cardioembolic stroke (P = .029). Plasma levels of soluble CD40L were significantly higher in patients with large artery disease compared to patients with cardioembolic stroke (P = .047). In conclusion, patients with acute ischemic stroke show an upregulation of platelet CD40L and an activation of cellular coagulation with highest activation in the large artery disease subgroup.