Simon F. Stämpfli

Guggulsterone, an anti-inflammatory phytosterol, inhibits tissue factor and arterial thrombosis

BackgroundThe phytosterol guggulsterone is a potent anti-inflammatory mediator with less side effects than classic steroids. This study assesses the impact of guggulsterone on tissue factor (TF) expression and thrombus formation.Methods and resultsGuggulsterone inhibited TNF-α-induced endothelial TF protein expression and surface activity in a concentration-dependent manner; in contrast, dexamethasone did not affect TNF-α-induced TF expression. Guggulsterone enhanced endothelial tissue factor pathway inhibitor and impaired plasminogen activator inhibitor-1 as well as vascular cell adhesion molecule-1 protein. Real-time polymerase chain reaction revealed that guggulsterone inhibited TNF-α-induced TF mRNA expression; moreover, it impaired activation of the MAP kinases JNK and p38, while that of ERK remained unaffected. In vivo, guggulsterone inhibited TF activity and photochemical injury induced thrombotic occlusion of mouse carotid artery. Guggulsterone also inhibited TF expression, proliferation, and migration of vascular smooth muscle cells in a concentration-dependent manner.ConclusionsGuggulsterone inhibits TF expression in vascular cells as well as thrombus formation in vivo; moreover, it impairs vascular smooth muscle cell activation. Hence, this phytosterol offers novel therapeutic options, in particular in inflammatory diseases associated with an increased risk of thrombosis.

Amiodarone Inhibits Arterial Thrombus Formation and Tissue Factor Translation

Background—In patients with coronary artery disease and reduced ejection fraction, amiodarone reduces mortality by decreasing sudden death. Because the latter may be triggered by coronary artery thrombosis as much as ventricular arrhythmias, amiodarone might interfere with tissue factor (TF) expression and thrombus formation. Methods and Results—Clinically relevant plasma concentrations of amiodarone reduced TF activity and impaired carotid artery thrombus formation in a mouse photochemical injury model in vivo. PTT, aPTT, and tail bleeding time were not affected; platelet number was slightly decreased. In human endothelial and vascular smooth muscle cells, amiodarone inhibited tumor necrosis factor (TNF)-&agr; and thrombin-induced TF expression as well as surface activity. Amiodarone lacking iodine and the main metabolite of amiodarone, N-monodesethylamiodarone, inhibited TF expression. Amiodarone did not affect mitogen-activated protein kinase activation, TF mRNA expression, and TF protein degradation. Metabolic labeling confirmed that amiodarone inhibited TF protein translation. Conclusions—Amiodarone impairs thrombus formation in vivo; in line with this, it inhibits TF protein expression and surface activity in human vascular cells. These pleiotropic actions occur within the range of amiodarone concentrations measured in patients, and thus may account at least in part for its beneficial effects in patients with coronary artery disease.

Laminin receptor activation inhibits endothelial tissue factor expression

Tissue factor (TF) is an important trigger of arterial thrombosis. The green tea catechin epigallocatechin-3-gallate (EGCG) is a ligand of the 67-kDa laminin receptor (67LR) and exhibits cardioprotective effects. This study investigates whether 67LR regulates TF expression in human endothelial cells. Immunofluorescence demonstrated that human aortic endothelial cells expressed 67LR. Cells grown on laminin expressed 35% less TF in response to TNF-alpha (TNF-alpha) than those grown on fibronectin (n=6; p<0.001). EGCG (1-30 microM) inhibited TNF-alpha and histamine induced endothelial TF expression and activity in a concentration dependent manner resulting in 87% reduction of TF expression (n=5; p<0.001); in contrast, expression of tissue factor pathway inhibitor was not affected (n=4; p=NS). In vivo administration of EGCG (30 mg/kg/day) inhibited TF activity in carotid arteries of C57BL6 mice. Real-time PCR and promoter studies revealed that EGCG decreased TF expression at the transcriptional level and impaired activation of the mitogen activated protein (MAP) kinase JNK 1/2, but not ERK or p38. Similarly, the JNK 1/2 inhibitor SP600125 (1 microM) impaired TF promoter activity (n=4; p<0.001) and protein expression (n=4; p<0.001). 67LR blocking antibodies blunted the inhibitory effect of EGCG on both TF protein expression and JNK activation. In contrast, vascular cell adhesion molecule 1 (VCAM-1) was not affected by laminin nor EGCG, and its expression was not regulated by JNK. EGCG did not affect TNF-alpha stimulated NFkB activation. Laminin receptor activation inhibits endothelial TF expression by impairing JNK phosphorylation. Thus, 67LR may be a potential target for the development of novel anti-thrombotic therapies.

Aging Induces Endothelial Dysfunction While Sparing Arterial Thrombosis

Objective—To assess the effects of aging on arterial thrombus formation by comparing 2-year-old with 11-week-old C57Bl6 mice. Methods and Results—Aging is a major risk factor for cardiovascular disease. In humans, assessing the direct effects of aging on vascular homeostasis is difficult because it occurs in the presence of other risk factors. Arterial thrombosis is the critical event in cardiovascular diseases; however, it is not known whether aging per se promotes its occurrence. Mice represent an interesting system to address this issue because they age without spontaneously developing other risk factors. Organ chamber experiments confirmed the advanced level of aging of old mice. As previously shown, old mice exhibited endothelial dysfunction; however, arterial thrombosis induced by photochemical injury was unchanged. Arterial tissue factor expression and activity; expressions of tissue factor pathway inhibitor, thrombomodulin, and plasminogen activator inhibitor 1; prothrombin time; partial thromboplastin time; thrombin-antithrombin complex; and platelet activation were comparable in both groups. Conclusion—Although these results cannot be directly extrapolated to humans, this study contributes novel important information on the direct effect of aging on arterial thrombosis and underscores the importance of controlling modifiable risk factors in aged individuals.

Restraint stress enhances arterial thrombosis in vivo - role of the sympathetic nervous system

Abstract Stress is known to correlate with the incidence of acute myocardial infarction. However, the molecular mechanisms underlying this correlation are not known. This study was designed to assess the effect of experimental stress on arterial thrombus formation, the key event in acute myocardial infarction. Mice exposed to 20 h of restraint stress displayed an increased arterial prothrombotic potential as assessed by photochemical injury-induced time to thrombotic occlusion. This increase was prevented by chemical sympathectomy performed through 6-hydroxydopamine (6-OHDA). Blood-born tissue factor (TF) activity was enhanced by stress and this increase could be prevented by 6-OHDA treatment. Vessel wall TF, platelet count, platelet aggregation, coagulation times (PT, aPTT), fibrinolytic system (t-PA and PAI-1) and tail bleeding time remained unaltered. Telemetric analysis revealed only minor hemodynamic changes throughout the stress protocol. Plasma catecholamines remained unaffected after restraint stress. Tumor necrosis factor alpha (TNF-α) plasma levels were unchanged and inhibition of TNF-α had no effect on stress-enhanced thrombosis. These results indicate that restraint stress enhances arterial thrombosis via the sympathetic nervous system. Blood-borne TF contributes, at least in part, to the observed effect whereas vessel wall TF, platelets, circulating coagulation factors, fibrinolysis and inflammation do not appear to play a role. These findings shed new light on the understanding of stress-induced cardiovascular events.

Increased prothrombotic profile in the left atrial appendage of atrial fibrillation patients

BACKGROUND Atrial fibrillation (AF) is associated with an increased risk for thromboembolic events. While observational data demonstrated that the majority of clots are formed within the left atrial appendage, the mechanisms behind this finding remain unclear also due to the fact that vitro studies so far have been hampered by the inability to isolate and culture cells from the atrial appendages. METHODS Patients suffering from AF undergoing cardiac surgery were recruited for this study and endocardial cells from their left (LAA) and right atrial appendage (RAA) were isolated and cultured according to a novel established protocol. Once in culture, cells were stimulated with TNF-α (10 ng/mL) and the expression of prothrombotic as well as proinflammatory markers was analyzed. RESULTS FACS analysis confirmed a high purity (98%) of isolated LAA endocardial cells. TNF-α significantly increased tissue factor (TF) and PAI-1 expression (n=5; P<0.005), while TFPI remained unchanged. Similarly, expression of VCAM-1 was significantly higher in the LAA as compared to the RAA (n=5; P<0.0001). CONCLUSION According to our newly established cell isolation protocol, this study reveals that in patients with AF, the endocardium of the LAA displays an increased prothrombotic and proinflammatory profile as compared to the RAA. This novel observation may constitute an important mechanism to explain the increased propensity of the LAA for clot formation, as well as the predominance of LAA-related thromboembolic complications in AF patients, and may have important implications for the development of novel treatment strategies.

Prognostic value of aortic regurgitation after TAVI in patients with chronic kidney disease

BACKGROUND Aortic regurgitation (AR) after transcatheter aortic valve implantation (TAVI) for severe aortic valve stenosis results in major haemodynamic changes. Influence of post-implant AR and aortic valve calcification on outcome in patients with chronic kidney disease (CKD) is unclear. METHODS Short-term outcome was defined as a combined 30-day endpoint, long-term outcome as survival. Post-implant AR was classified as none/mild or moderate/severe using transthoracic echocardiography. Aortic valve calcification was calculated by computed tomography. Logistic regression analyses were performed in patients with none/mild (estimated glomerular filtration rate [eGFR]≥30ml/min/1.73m(2)) and advanced (eGFR<30ml/min/1.73m(2)) CKD to evaluate predictors of outcome and post-implant AR. RESULTS TAVI was performed in 546 consecutive patients. Moderate/severe post-implant AR was the only independent predictor of the 30-day endpoint in patients with advanced (OR 7.091, 95% CI 1.144-43.962, p=0.035), but not in patients with none/mild CKD. Similarly, moderate/severe AR predicted impaired survival only in patients with advanced CKD (p<0.001). NT-proBNP (OR 1.023 per 500ng/l increase, 95% CI 1.003-1.043; p=0.026) before intervention was the only independent predictor of the 30-day endpoint in patients with none/mild CKD. Aortic valve calcification was comparable in patients with none/mild versus advanced CKD and was an independent predictor of moderate/severe post-implant AR in the overall population as well as in the subgroups with none/mild or advanced CKD. CONCLUSIONS Moderate/severe AR after TAVI predicts outcome in patients with advanced CKD, but not in patients with none/mild CKD. Aortic valve calcification is an important predictor of post-implant AR independent of kidney function.

Haemosiderin-Laden Sputum Macrophages for Diagnosis in Pulmonary Veno-Occlusive Disease

Aims Pulmonary veno-occlusive disease (PVOD) is a rare condition of pulmonary arterial hypertension (PAH), in which post-capillary veins are affected. Since the therapeutic approach in PVOD differs from other forms of PAH, it is crucial to establish the diagnosis. Due to the fact that affected patients are often hemodynamically unstable, minimal invasive procedures are necessary for the diagnostic work-up. Chronic alveolar haemorrhage has been observed during bronchoalveolar lavage in PVOD cases. This study therefore investigates whether signs of alveolar haemorrhage can also be found in the sputum of these patients. Methods and Results Six patients suffering from PVOD were included in this analysis. As controls, patients with idiopathic PAH (n = 11), chronic thromboembolic PH (n = 9) and with sclerodermia-associated PH (n = 10) were assessed. Sputum from every patient was obtained by a non-invasive manner. The amount of haemosiderin-laden macrophages was determined using the Golde score. There were statistically significant more haemosiderin-laden macrophages in the sputum of patients suffering from PVOD as compared to the other groups (P<0.05). Assuming a cut-off of 200 on the Golde score, all of the 6 PVOD patients surpassed this value compared with only 1 out of the 30 cases with precapillary PH. Thus, sensitivity and specificity with respect to the diagnosis of PVOD was 100% and 97%, respectively. Conclusion The content of haemosiderin-laden macrophages in the sputum of patients suffering from PVOD is significantly higher as compared to other forms of PH and may be useful in the non-invasive diagnostic work-up of these patients.

Ticagrelor but not clopidogrel active Metabolite displays antithrombotic Properties in the left Atrial Endocardium

Aims Oral anticoagulation is considered standard therapy for stroke prevention in atrial fibrillation (AF). Endocardial activation triggers expression of pro-thrombotic mediators including tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1), and contributes to thrombus formation in the left atrial appendage (LAA) of AF patients. Recently, pleiotropic effects of specific P2Y12 receptor antagonists were demonstrated; however, whether these drugs possess antithrombotic effects on LAA endocardial cells currently remains unknown. Methods and results LAA were obtained from 14 patients with known AF undergoing elective cardiac surgery including LAA removal at the University Hospital Zurich. LAA endocardial cells were isolated and pre-incubated with ticagrelor (10-7, 10-6, 10-5M) or clopidogrel active metabolite (CAM) (1.5 × 10-8, 1.5 × 10-7, 1.5 × 10-6 M) before stimulation with tumour necrosis factor-alpha (TNF-α) (10 ng/mL). Finally, TF and PAI-1 expression and activity were analysed. Ticagrelor, unlike CAM, concentration dependently decreased TNF-α-induced TF expression and TF activity in LAA endocardial cells. Further, ticagrelor, but not CAM reduced PAI-1 expression and enzyme activity in TNF-α-stimulated LAA endocardial cells. In contrast, TF pathway inhibitor (TFPI) remained unaffected by both dugs. Conclusion Ticagrelor, but not CAM, reduces expression and activity of TF and PAI-1 in LAA endocardial cells isolated from patients with AF, indicating possible local antithrombotic effects. Such pleiotropic properties of ticagrelor may contribute to a reduction in thromboembolic complications in patients with AF.

Caffeine induces endothelial tissue factor expression via phosphatidylinositol 3-kinase inhibition

Tissue factor (TF) is the key activator of coagulation and is involved in acute coronary syndromes. Caffeine is often reported to increase cardiovascular risk; however, its effect on cardiovascular morbidity and mortality is controversial. Hence, this study was designed to investigate the impact of caffeine on endothelial TF expression in vitro. Caffeine concentration-dependently enhanced TF protein expression and surface activity in human endothelial cells stimulated by tumour necrosis factor (TNF)-α or thrombin. Caffeine inhibited phosphatidylinositol 3-kinase (PI3K) activity and this effect was comparable to that of the known PI3K inhibitor LY294002. Consistently, treatment of endothelial cells with LY294002 enhanced TNF-α induced TF expression to a similar extent as caffeine, and adenoviral expression of the active PI3K mutant (p110) reversed the effect of both caffeine and LY294002 on TF expression. Caffeine and LY294002 increased DNA binding capacity of the transcription factor nuclear factor κB, whereas the activation pattern of mitogen-activated protein kinases (MAPK) remained unaltered. Luciferase reporter assay revealed a caffeine dependent activation of the TF promoter, and RT-PCR revealed a dose dependent increase in TF mRNA levels when stimulated with caffeine in the presence of TNF-α. In conclusion, caffeine enhances TNF-α-induced endothelial TF protein expression as well as surface activity by inhibition of PI3K signalling. Since the caffeine concentrations applied in the present study are within the plasma range measured in humans, our findings indicate that caffeine enhances the prothrombotic potential of endothelial cells and underscore the importance of PI3K in mediating these effects.