Elif Elmas

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.

Long-term prognostic value of midregional pro-adrenomedullin and C-terminal pro-endothelin-1 in patients with acute myocardial infarction

Abstract Background: Midregional pro-adrenomedullin (MR-proADM) and endothelin-1 have been shown to predict mortality of patients with acute myocardial infarction. However, the prognostic value of both biomarkers in predicting long-term clinical events after acute myocardial infarction remains unclear. Methods: In a prospective study, 30 patients suffering from acute ST elevation myocardial infarction or non-ST elevation myocardial infarction were enrolled. Measurements of MR-proADM and CT-pro-endothelin-1 (CT-proET-1) were performed at initial presentation, 2 or 3 days and 4 months after acute myocardial infarction. Long-term clinical events (e.g., recurrent myocardial infarction, percutaneous transluminal coronary angioplasty, aorto-coronary venous bypass or cardiogenic shock) were documented over a period from the 4th until the 10th month. Results: Both MR-proADM and CT-proET-1 were able to differentiate patients with subsequent long-term clinical events (n=11) from those without (n=19). At the time of acute myocardial infarction, median MR-proADM level of the event group was 0.69 nmol/L as compared to 0.59 nmol/L of the no-event group (p=0.036). A difference was still observed after 3 days (event group median 0.66 nmol/L; no-event group median 0.57 nmol/L; p=0.022). Accordingly, median CT-proET-1 level was 72.9 pmol/L in the event group as compared to a median of 54.4 pmol/L in patients in the no-event group (p=0.009) 3 days after acute myocardial infarction. Within the acute phase, patients with MR-proADM levels ≥0.67 nmol/L were 3 times more likely (relative risk 2.8; 95% confidence interval 1.2–6.9; p=0.042) to suffer from a future clinical event. The area under the curve (AUC) was 0.71 (95% confidence interval 0.51–0.86; p=0.046). After 3 days, patients with CT-proET-1 levels ≥57 pmol/L were 6 times more likely (relative risk 5.9; 95% confidence interval 0.9–40.4; p=0.036) to suffer from a future clinical event. The AUC was 0.76 (95% confidence interval 0.55–0.90; p=0.015). Conclusions: Elevated levels of MR-proADM and CT-proET-1 during the acute phase of myocardial infarction may predict an adverse long-term clinical outcome. Clin Chem Lab Med 2008;46:204–11.

Characteristics and long-term outcome of right ventricular involvement in Takotsubo cardiomyopathy

OBJECTIVE Takotsubo cardiomyopathy, also known as stress-induced cardiomyopathy (SCM) resembles a reversible cardiomyopathy that is characterized by localized wall motion abnormalities in the absence of stenotic coronary vascular disease. Patients typically present with apical ballooning of the left ventricle (LV), however the right ventricle (RV) is also affected in up to 50.0% of patients. Long-term prognosis of classical SCM resembles that of patients after ST elevation myocardial infarction. Data on long-term prognosis of biventricular compared to classical SCM is controversial. The aim of this study was therefore to analyze patients with biventricular SCM regarding in-hospital outcome and long-term prognosis. MATERIALS AND METHODS 114 consecutive patients with SCM were retrospectively analyzed. 88 patients presented with classical SCM, 26 patients (22.8%) were diagnosed with biventricular SCM. Follow-up was conducted for a total of 4.4years. Mean age was 67.1years with 83.3% of patients being female. The primary endpoint was a composite of all-cause mortality, recurrence of SCM and re-hospitalization due to heart failure. RESULTS Although patients with biventricular SCM presented with a tendency towards an increased rate of cardiogenic shock (30.8% vs. 15.9%; p=0.09) and significantly more usage of inotropic support upon hospital admission (34.6% vs. 13.6%; p=0.01), there was no difference concerning the primary endpoint in both groups (50.0% vs. 44.3%; p=0.31). Furthermore, there was no difference in mortality both in-hospital (7.7% vs. 7.9%; p=0.66) and during long-term follow-up (27.3% vs. 23.1%; p=0.46). CONCLUSION Patients with biventricular SCM have the same in-hospital and long-term outcome compared to classical SCM.

Spectroscopic diagnosis of myocardial infarction and heart failure by Fourier transform infrared spectroscopy in serum samples.

Cardiovascular disease is the leading cause of death in Western civilization. In this pilot study we evaluated a new method for the diagnosis of myocardial infarction and heart failure by determining the typical fingerprint in the infrared (IR) spectrum of 1 μL of a dried patient serum sample by Fourier transform IR spectroscopy. For classification, cluster analysis and artificial neural networks (ANN) were applied. In this study 567 subjects were enrolled, comprising 225 controls (Co) and 342 patients with myocardial infarction (MI) (n = 157) and heart failure (HF) (n = 185). By applying artificial neural network algorithms, the following sensitivities and specificities of the same spectra were determined: MI versus Co (98%, 97%), HF versus Co (98%, 100%), MI versus HF (100%, 100%), and MI plus HF versus Co (100%, 100%). Based on our data, mid-IR spectroscopy appears to be a promising new method to diagnose heart diseases from serum samples. Artificial neural network algorithms proved to be superior to cluster analysis for correct prediction.

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.

Impact of concomitant atrial fibrillation on the prognosis of Takotsubo cardiomyopathy

Aims Previous studies revealed that patients with Takotsubo cardiomyopathy (TTC) have a higher mortality rate than the general population. Supraventricular tachycardia is a well-known complication of TTC. This study was performed to determine the short- and long-term prognostic impact of atrial fibrillation associated with TTC patients. Methods and results Our institutional database constituted a collective of 114 patients diagnosed with TTC from 2003 to 2015. The patients were divided into two groups according to the presence (n = 21, 18.4%) or absence (n = 93, 81.5%) of atrial fibrillation. The endpoint was a composite of in-hospital events (thromboembolic events and life-threatening arrhythmias), all-cause mortality, rehospitalization due to heart failure, stroke, and the recurrence of TTC. The in-hospital mortality, 30-day mortality, and long-term mortality were significantly higher in the atrial fibrillation group. Kaplan-Meier analysis indicated a significantly lower event-free survival rate over a mean follow-up of 3 years in the atrial fibrillation group than that in the non-atrial fibrillation group (log-rank, P < 0.01). In a multivariate cox regression analysis, atrial fibrillation (hazard ratio, HR 2.3, 95% confidence interval, CI: 1.1-4.9, P < 0.05) and EF ≤ 35% (HR 2.0, 95% CI: 1.1-3.8, P < 0.05) were the only independent predictors of a primary endpoint. Conclusion Rates of in-hospital events and short- as well as long-term mortality were significantly higher in TTC patients suffering from atrial fibrillation compared with patients without atrial fibrillation.