Alexander Breitenstein

Sirt1 inhibition promotes in vivo arterial thrombosis and tissue factor expression in stimulated cells

AIMS The mammalian silent information regulator-two 1 (Sirt1) blunts the noxious effects of cardiovascular risk factors such as type 2 diabetes mellitus and obesity. Nevertheless, the role of Sirt1 in regulating the expression of tissue factor (TF), the key trigger of coagulation, and arterial thrombus formation remains unknown. METHODS AND RESULTS Human as well as mouse cell lines were used for in vitro experiments, and C57Bl/6 mice for in vivo procedures. Sirt1 inhibition by splitomicin or sirtinol enhanced cytokine-induced endothelial TF protein expression as well as surface activity, while TF pathway inhibitor protein expression did not change. Sirt1 inhibition further enhanced TF mRNA expression, TF promoter activity, and nuclear translocation as well as DNA binding of the p65 subunit of nuclear factor-kappa B (NFκB/p65). Sirt1 siRNA enhanced TF protein and mRNA expression, and this effect was reduced in NFκB/p65(-/-) mouse embryonic fibroblasts reconstituted with non-acetylatable Lys(310)-mutant NFκB/p65. Activation of the mitogen-activated protein kinases p38, c-Jun NH(2)-terminal kinase, and p44/42 (ERK) remained unaffected. In vivo, mice treated with the Sirt1 inhibitor splitomicin exhibited enhanced TF activity in the arterial vessel wall and accelerated carotid artery thrombus formation in a photochemical injury model. CONCLUSION We provide pharmacological and genetic evidence that Sirt1 inhibition enhances TF expression and activity by increasing NFκB/p65 activation in human endothelial cells. Furthermore, Sirt1 inhibition induces arterial thrombus formation in vivo. Hence, modulation of Sirt1 may offer novel therapeutic options for targeting thrombosis.

Rapamycin promotes arterial thrombosis in vivo: implications for everolimus and zotarolimus eluting stents

AIMS Drug-eluting stents (DES) may be associated with an increased risk for stent thrombosis when compared with bare-metal stents. In endothelial cells, rapamycin induces tissue factor (TF) by inhibiting the mammalian target of rapamycin (mTOR). However, the effect of mTOR inhibition on TF activity and thrombus formation in vivo has not yet been studied. Moreover, it is unclear whether second-generation DES substances everolimus and zotarolimus have an effect on endothelial TF expression. METHODS AND RESULTS In a mouse carotid artery photochemical injury model, rapamycin (182 +/- 27.5 microg/L) decreased time to thrombotic occlusion by 40%, increased TF activity, and abrogated p70S6K phosphorylation when compared with controls. In vitro, rapamycin, everolimus, and zotarolimus (each 10(-7) mol/l) enhanced TNF-alpha-induced TF expression by 2.2-, 1.7-, and 2.4-fold, respectively, which was paralleled by an increase in TF surface activity. Similar to rapamycin, everolimus and zotarolimus abrogated TNF-alpha-induced p70S6K phosphorylation under these conditions. CONCLUSION Rapamycin increases TF activity and promotes arterial thrombosis in vivo at concentrations relevant in patients undergoing DES implantation; this effect may increase the thrombogenicity of DES. Since everolimus and zotarolimus augment endothelial TF expression and activity in vitro in a similar manner as rapamycin, these findings may also be relevant for second generation DES.

Tissue factor: beyond coagulation in the cardiovascular system

TF (tissue factor) is the main trigger of the coagulation cascade; by binding Factor VIIa it activates Factor IX and Factor X, thereby resulting in fibrin formation. Various stimuli, such as cytokines, growth factors and biogenic amines, induce TF expression and activity in vascular cells. Downstream targets of these mediators include diverse signalling molecules such as MAPKs (mitogen-activated protein kinases), PI3K (phosphoinositide 3-kinase) and PKC (protein kinase C). In addition, TF can be detected in the bloodstream, known as circulating or blood-borne TF. Many cardiovascular risk factors, such as hypertension, diabetes, dyslipidaemia and smoking, are associated with increased expression of TF. Furthermore, in patients presenting with acute coronary syndromes, elevated levels of circulating TF are found. Apart from its role in thrombosis, TF has pro-atherogenic properties, as it is involved in neointima formation by inducing vascular smooth muscle cell migration. As inhibition of TF action appears to be an attractive target for the treatment of cardiovascular disease, therapeutic strategies are under investigation to specifically interfere with the action of TF or, alternatively, promote the effects of TFPI (TF pathway inhibitor).

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.

Peripheral blood monocyte Sirt1 expression is reduced in patients with coronary artery disease.

Background Inflammation plays a key role in atherosclerosis. Sirt1 regulates transcription factors involved in inflammatory processes and blunts atherosclerosis in mice. However, its role in humans remains to be defined. This study was therefore designed to investigate the role of Sirt1 in the development of atherosclerosis. Methods and Results 48 male subjects admitted for cardiac catheterization were subdivided into healthy subjects, patients with stable coronary artery disease (CAD), and with acute coronary syndromes (ACS). Monocytes were isolated and Sirt1 mRNA levels were determined. Sirt1 gene expression was higher in healthy subjects as compared to patients with CAD or ACS (P<0.05), respectively. Interestingly, HDL levels correlated positively with Sirt1 expression. Thus, HDL from the three groups was isolated and incubated with THP-1 monocytes to determine the effects of HDL on Sirt1 protein in controlled experimental conditions. HDL from healthy subjects stimulated Sirt1 expression in THP-1 monocytes to a higher degree than HDL from CAD and ACS patients (P<0.05). Paraoxonase-1 (PON-1), a HDL-associated enzyme, showed a reduced activity in HDL isolated from CAD and ACS patients as compared to the controls (P<0.001). Conclusions Monocytic Sirt1 expression is reduced in patients with stable CAD and ACS. Experiments on THP-1 monocytes suggest that this effect is HDL-dependent and is mediated by a reduced activity of HDL-associated enzyme PON1.

Endothelial overexpression of LOX-1 increases plaque formation and promotes atherosclerosis in vivo.

AIMS Lectin-like oxLDL receptor-1 (LOX-1) mediates the uptake of oxidized low-density lipoprotein (oxLDL) in endothelial cells and macrophages. However, the different atherogenic potential of LOX-1-mediated endothelial and macrophage oxLDL uptake remains unclear. The present study was designed to investigate the in vivo role of endothelial LOX-1 in atherogenesis. METHODS AND RESULTS Endothelial-specific LOX-1 transgenic mice were generated using the Tie2 promoter (LOX-1TG). Oxidized low-density lipoprotein uptake was enhanced in cultured endothelial cells, but not in macrophages of LOX-1TG mice. Six-week-old male LOX-1TG and wild-type (WT) mice were fed a high-cholesterol diet (HCD) for 30 weeks. Increased reactive oxygen species production, impaired endothelial nitric oxide synthase activity and endothelial dysfunction were observed in LOX-1TG mice as compared with WT littermates. LOX-1 overexpression led to p38 phosphorylation, increased nuclear factor κB activity and subsequent up-regulation of vascular cell adhesion molecule-1, thereby favouring macrophage accumulation and aortic fatty streaks. Consistently, HCD-fed double-mutant LOX-1TG/ApoE(-/-) displayed oxidative stress and vascular inflammation with higher aortic plaques than ApoE(-/-) controls. Finally, bone marrow transplantation experiments showed that endothelial LOX-1 was sufficient for atherosclerosis development in vivo. CONCLUSIONS Endothelial-specific LOX-1 overexpression enhanced aortic oxLDL levels, thereby favouring endothelial dysfunction, vascular inflammation and plaque formation. Thus, LOX-1 may serve as a novel therapeutic target for atherosclerosis.

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.

Cardiac Glycosides Regulate Endothelial Tissue Factor Expression in Culture

Background—Tissue factor (TF) plays an important role in acute coronary syndromes and stent thrombosis. This study investigates whether Na+/K+-ATPase regulates TF expression in human endothelial cells. Methods and Results—Ouabain inhibited tumor necrosis factor (TNF)-&agr;–induced endothelial TF protein expression; maximal inhibition occurred at 10−5 mol/L, reached more than 70%, and was observed throughout the 5 hours stimulation period. The decrease in protein expression was paralleled by a reduced TF surface activity. Similarly, lowering of extracellular potassium concentration inhibited TNF-&agr;–induced TF protein expression. In contrast, ouabain did not affect TNF-&agr;–induced expression of full-length TF mRNA for up to 5 hours of stimulation; instead, expression of alternatively-spliced TF mRNA was upregulated after 3 and 5 hours of stimulation. Ouabain did not affect TNF-&agr;–induced activation of the MAP kinases p38, extracellular signal-regulated kinase (ERK), and c-Jun terminal NH2 kinase; activation of Akt and p70S6 kinase remained unaltered as well. Similar to the MAP kinases, ouabain did not affect TNF-&agr;–induced degradation of I&kgr;B-&agr;. Ouabain had no effect on TF protein degradation. Conclusions—Na+/K+-ATPase is required for protein translation of endothelial TF in culture. This observation provides novel insights into posttranscriptional regulation of TF expression.

Amphetamines induce tissue factor and impair tissue factor pathway inhibitor: role of dopamine receptor type 4

AIMS Amphetamine intake is associated with acute vascular syndromes. Since these events are caused by arterial thrombosis and this in turn is triggered by tissue factor (TF), this study examines whether amphetamines regulate TF in human endothelial cells. METHODS AND RESULTS Amphetamine (10(-7)-10(-4) mol/L) enhanced thrombin- and tumour necrosis factor (TNF)-alpha-induced as well as basal TF expression (P = 0.029, 0.0003, and 0.003 at maximal concentration), and TNF-alpha-induced plasminogen activator inhibitor (PAI)-1 expression (P = 0.003), whereas tissue factor pathway inhibitor expression was impaired (P = 0.008). Similarly, 3,4-methylenedioxymethamphetamine (10(-7)-10(-4) mol/L) enhanced TF expression (P = 0.046). These effects were paralleled by an increased TF activity (P = 0.002); moreover, clotting time of human plasma was accelerated by supernatant from amphetamine-treated cells (P = 0.03). Amphetamine enhanced TF mRNA expression via phosphorylation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK) and p38 (P = 0.03 and 0.033), but not c-Jun NH(2)-terminal kinase (JNK; P = 0.81). The effect of amphetamine on TF expression was abrogated by the dopamine D4 receptor antagonists L-745,870 and L-750,667, but not D2 or D3 receptor antagonists; furthermore, L-745,870 blunted the amphetamine-induced activation of ERK and p38, but not JNK. CONCLUSION Amphetamines induce endothelial TF expression via stimulation of dopamine D4 receptor and activation of the MAPKs p38 and ERK. These effects occur at clinically relevant amphetamine concentrations and may account for the increased incidence of acute vascular syndromes after amphetamine consumption.

Endothelial LOX-1 activation differentially regulates arterial thrombus formation depending on oxLDL levels: role of the Oct-1/SIRT1 and ERK1/2 pathways

Aims The lectin-like oxLDL receptor-1 (LOX-1) promotes endothelial uptake of oxidized low-density lipoprotein (oxLDL) and plays an important role in atherosclerosis and acute coronary syndromes (ACS). However, its role in arterial thrombus formation remains unknown. We investigated whether LOX-1 plays a role in arterial thrombus formation in vivo at different levels of oxLDL using endothelial-specific LOX-1 transgenic mice (LOX-1TG) and a photochemical injury thrombosis model of the carotid artery. Methods and results In mice fed a normal chow diet, time to arterial occlusion was unexpectedly prolonged in LOX-1TG as compared to WT. In line with this, tissue factor (TF) expression and activity in carotid arteries of LOX-1TG mice were reduced by half. This effect was mediated by activation of octamer transcription factor 1 (Oct-1) leading to upregulation of the mammalian deacetylase silent information regulator-two 1 (SIRT1) via binding to its promoter and subsequent inhibition of NF-&kgr;B signaling. In contrast, intravenous injection of oxLDL as well as high cholesterol diet for 6 weeks led to a switch from the Oct-1/SIRT1 signal transduction pathway to the ERK1/2 pathway and in turn to an enhanced thrombotic response with shortened occlusion time. Conclusions Thus, LOX-1 differentially regulates thrombus formation in vivo depending on the degree of activation by oxLDL. At low oxLDL levels LOX-1 activates the protective Oct-1/SIRT1 pathway, while at higher levels of the lipoprotein switches to the thrombogenic ERK1/2 pathway. These findings may be important for arterial thrombus formation in ACS and suggest that SIRT1 may represent a novel therapeutic target in this context.