Regulation of tissue factor expression: implications for coronary artery disease

Das Endothel als innerste Schicht eines Blutgefässes stellt eine Verbindung zwischen dem Blutstrom und dem Rest der Gefässwand dar. Es fungiert als eine physikalische Barriere zwischen Blut und Gefäss, dient als Quelle für Wachstumsfaktoren –und Inhibitoren sowie für Enzyme, die kardiovaskuläre Hormone sowohl aktivieren als auch deaktivieren, und ist an der Produktion von Kontraktions- bzw. Relaxations-Faktoren beteiligt. Risikofaktoren wie Daibetes, Hypertonie, Adipositas und Rauchen stören die Integrität des Endothels und führen zum Verlust der Funktionalität. Eine endotheliale Dysfunktion wird durch eine reduzierte NO Synthese gekennzeichnet, was wiederum die Aufnahme und Oxidierung zirkulierender Lipoproteine und Monozyten durch das Endothel in die Intima erleichtert. Diese vortschreitende prozess führt möglicherweise zu einer frühen arteriosklerotischen Läsion, welche sich mit der Zeit zu einer plaque mit nekrotischem Kern und fibroider Schale entwickelt, woraus im weiteren Verlauf eine plaque mit Gefahr der Ruptur entstehen kann. Die Ruptur einer instabilen Plaque ist verantwortlich für koronare Thromben, die Hauptursachen für ein akutes coronares Syndrom. Perkutane transluminale koronare Angioplastie ist ein in der Klinik routinemässig eingesetztes Verfahren zur Revaskularisierung verschlossener Gefässe. Daran angeschlossen ist die Einlage einer als Stent bezeichneten röhrenförmigen Struktur, was das wiedereröffnete Gefäss vor erneutem Verschluss bewahren soll. Stents sind gewöhnlich mit Substanzen beschichtet, welche die für eine Restenose ursächliche excessive Proliferation glatter Gefässmuskulatur unterbindet. Wie neulich gezeigt werden konnte ist, im Gegensatz zu Restenose, die Inzidenz für In-Stent-Thrombosen durch die Anwendung von sogenannten „Drug eluting stents“ nicht gesunken. Es konnte nachgewiesen werden, dass das vielfach zur Beschichtung von Stents verwendete Rapamycin die Expression von TF, Hauptinitiator der Koagulationskaskade, induziert, was ein neues Licht auf mögliche Ursachen für das Auftreten von In-Stent- Thrombosen wirft. Im ersten teil dieser Arbeit charakterisieren wir die Auswirkungen des als alternativ zu Rapamycin vorwiegend verwendeten Paclitaxel auf die TFExpresssion in menschlichen Endothelzellen. In der Tat steigert Paclitaxel die durch Thrombin induzierte endotheliale TF Protein Expression sowohl konzentrations-als auch zeitabhängig. Eine Konzentration von 10-5 mol/l erbrachte eine 2,1-fache Erhöhung in Bezug auf TF Protein und einen 1,6-fachen Anstieg der TF Oberflächenaktivität. Die Prästimulation für 1h ergab im Vergleich zu einer Vorbehandlung für 25 h keinen wesentlichen Unterschied. Anhand RT-PCR konnte gezeigt werden, dass Paclitaxel die durch Thrombin induzierte TF mRNA Expression ebenfalls erhöht. Darüber hinaus potenzierte Paclitaxel die Aktivierung der c-Jun terminalen NH2 Kinase (JNK) durch Thrombin, wohingegen die durch Thrombin vermittelte Phosphorilierung von p38 und der extrazellulären signal-regulierten Kinase (ERK) unter Zugabe von Paclitaxel unverändert blieb. Docetaxol steigerte, ähnlich dem Paclitaxel, sowohl TF Expression als auch JNK Aktivierung im Vergleich zur alleinigen Applikation von Thrombin. Der JNK Inhibitor SP600125 konnte die Thrombin-induzierte TF Expression um 35 % reduzieren. Im weiteren wurde durch SP600125 der Effekt von sowohl Paclitaxel als auch Docetaxel bezüglich TF Expression verschleiert. Schwerpunkt des zweiten Teils der Arbeit war die Untersuchung von Dimethyl Sulfoxid (DMSO) als potentielle neue Substanz zur coating von Stents. DMSO wird zur Konservierung von hämapoetischen Stammzellen verwendet und Patienten vor Knochenmarktransplantation infundiert. Trotz seiner intravenösen Anwendung wurde die Auswirkung von DMSO auf vaskuläre Zellen noch nicht untersucht. In dieser Studie konnte gezeigt werden, dass DMSO die TF Expression in menschlichen Endothelzellen, Monozyten und VSMC unterdrückt. RT-PCR zeigte die Inhibition die TF Expression auf mRNA Ebene, was durch die reduzierte Aktivierung der MAP Kinasen JNK und p38, jedoch nicht ERK, vermittelt wurde. In vivo unterdrückt DMSO die TF Aktivität und verhinderte im Maus-Modell einen thrombotischen Verschluss. Darüber hinaus inhibierte DMSO konzentrationsabhängig die Proliferation und Migration von glatten Gefässmuskelzellen, vielmehr verhinderte es die Rapamycin bzw. Paclitaxel induzierte Hochregulation der TF Expression. Da DMSO auf unterschiedlichem Gebiet der modernen Medizin bereits etabliert zur Anwendung kommt, empfehlen wir diese Substanz als neuwertiges Verfahren zur Behandlung eines Akutem Koronarsyndrom; im speziellen, DMSO scheint ein attraktives Mittel zur Beschichtung von Drug-eluting Stents zu sein, entweder allein oder in Kombination mit Rapamycin oder Paclitaxel.The innermost layer of a vessel, the endothelium, forms an interface between the circulating blood in the vascular lumen and the vessel wall. The endothelium functions as a.) a physical barrier between the blood and the vessel; b.) a vital source of enzymes activating and deactivating cardiovascular hormones; c.) a site of production of relaxing and contracting factors; and d.) a source of growth inhibitors and promoters. Risk factors such as diabetes, hypertension, obesity and smoking can disturb the integrity of the endothelium rendering it dysfunctional. A dysfunctional endothelium is characterized by a decreased endotheliumdependent relaxation which in turn facilitates the entry and oxidation of circulating lipoproteins and monocytes through the endothelium into the intima. The advancing accumulation of lipoproteins and monocytes into the intima eventually gives rise to an early atherosclerotic lesion which later develops into a necrotic core and a fibrous cap and ultimately becomes an advanced plaque at risk of rupture. Rupture of unstable plaques is responsible for coronary thrombosis, the main cause of unstable angina, acute myocardial infarction, and sudden cardiac death. Percutaneous transluminal coronary angioplasty is a clinical procedure routinely employed to revascularise occluded vessels. Following percutaneous transluminal coronary angioplasty a tube-like metal structure, a stent, is inserted into the vessel to prevent it from occluding once again. Stents are commonly coated with drugs orientated at inhibiting the excessive proliferation of vascular smooth muscle responsible for restenosis. Unlike for restenosis, recent evidence has shown that the incidence of in stent thrombosis has not decreased following the advent of drug eluting stents. Rapamycin which is widely used for coating stents has been shown to induce the expression of TF, the key initiator of the coagulation cascade. This finding shed new light on the possible causes for the occurrence of stent thrombosis. In the first part of this thesis we characterised the impact of paclitaxel, the main alternative to rapamycin, on TF expression in human endothelial cells. Indeed, paclitaxel enhanced thrombin-induced endothelial TF protein expression in a concentration- and time-dependent manner. A concentration of 10-5 mol/L elicited a 2.1-fold increase in TF protein and a 1.6-fold increase in cell surface TF activity. The effect was similar after a 1 h as compared to a 25 h pretreatment period. Real-time polymerase chain reaction revealed that paclitaxel increased thrombin-induced TF mRNA expression. Paclitaxel potently activated c-Jun terminal NH2 kinase (JNK) as compared to thrombin alone, while the thrombin-mediated phosphorylation of p38 and extracellular signal-regulated kinase remained unaffected. Similar to paclitaxel, docetaxel enhanced both TF expression and JNK activation as compared to thrombin alone. The JNK inhibitor SP600125 reduced thrombin-induced TF expression by 35%. Moreover, SP600125 blunted the effect of paclitaxel and docetaxel on thrombin-induced TF expression. Paclitaxel increases endothelial TF expression via selective activation of JNK. This observation provides novel insights into the pathogenesis of thrombus formation after paclitaxel-eluting stent deployment and may have an impact on drug-eluting stent design. In the second part of this thesis we directed our efforts at characterising the potential application of Dimethyl sulfoxide (DMSO) as a novel agent to be used for stent coating. DMSO is used for preservation of hematopoietic progenitor cells and infused into patients undergoing bone marrow transplantation. Despite of its intravenous application, the impact of DMSO on vascular cells has not been assessed. In this study we found that DMSO inhibited TF expression in human endothelial cells, monocytes, and VSMC. Real-time PCR revealed that inhibition of TF expression occurred at the mRNA level. This effect was mediated by reduced activation of the MAP kinases JNK and p38, but not ERK. In vivo, DMSO treatment suppressed TF activity and prevented thrombotic occlusion in a mouse model of carotid artery photochemical injury. DMSO also inhibited VSMC proliferation and migration in a concentration-dependent manner; moreover, it prevented rapamycin and paclitaxel-induced upregulation of TF expression. As the use of DMSO is established in different areas of modern medicine, we propose this drug as a novel strategy for treating acute coronary syndromes; in particular, DMSO seems to represent an attractive compound for application on drug-eluting stents, either alone or in combination with rapamycin or paclitaxel

Molecular mechanism of endothelial and vascular aging: implications for cardiovascular disease

Western societies are aging due to an increasing life span, decreased birth rates, and improving social and health conditions. On the other hand, the prevalence of cardiovascular (CV) and cerebrovascular (CBV) diseases rises with age. Thus, in view of the ongoing aging pandemic, it is appropriate to better understand the molecular pathways of aging as well as age-associated CV and CBV diseases. Oxidative stress contributes to aging of organs and the whole body by an accumulation of reactive oxygen species promoting oxidative damage. Indeed, increased oxidative stress produced in the mitochondria and cytosol of heart and brain is a common denominator to almost all CV and CBV diseases. The mitochondrial adaptor protein p66Shc and the family of deacetylase enzymes, the sirtuins, regulate the aging process, determine lifespan of many species and are involved in CV diseases. GDF11, a member of TGFβ superfamily with homology to myostatin also retards the aging process via yet unknown mechanisms. Recent evidence points towards a promising role of this novel ‘rejuvenation' factor in reducing age-related heart disease. Finally, telomere length is also involved in aging and the development of age-related CV dysfunction. This review focuses on the latest scientific advances in understanding age-related changes of the CV and CBV system, as well as delineating potential novel therapeutic targets derived from aging research for CV and CBV disease

Cytokines as therapeutic targets for cardio- and cerebrovascular diseases

Despite major advances in prevention and treatment, cardiac and cerebral atherothrombotic complications still account for substantial morbidity and mortality worldwide. In this context, inflammation is involved in the chronic process leading atherosclerotic plaque formation and its complications, as well as in the maladaptive response to acute ischemic events. For this reason, modulation of inflammation is nowadays seen as a promising therapeutic strategy to counteract the burden of cardio- and cerebrovascular disease. Being produced and recognized by both inflammatory and vascular cells, the complex network of cytokines holds key functions in the crosstalk of these two systems and orchestrates the progression of atherothrombosis. By binding to membrane receptors, these soluble mediators trigger specific intracellular signaling pathways eventually leading to the activation of transcription factors and a deep modulation of cell function. Both stimulatory and inhibitory cytokines have been described and progressively reported as markers of disease or interesting therapeutic targets in the cardiovascular field. Nevertheless, cytokine inhibition is burdened by harmful side effects that will most likely prevent its chronic use in favor of acute administrations in well-selected subjects at high risk. Here, we summarize the current state of knowledge regarding the modulatory role of cytokines on atherosclerosis, myocardial infarction, and stroke. Then, we discuss evidence from clinical trials specifically targeting cytokines and the potential implication of these advances into daily clinical practice

Modern Concepts in Cardiovascular Disease: Inflamm-Aging

The improvements in healthcare services and quality of life result in a longer life expectancy and a higher number of aged individuals, who are inevitably affected by age-associated cardiovascular (CV) diseases. This challenging demographic shift calls for a greater effort to unravel the molecular mechanisms underlying age-related CV diseases to identify new therapeutic targets to cope with the ongoing aging "pandemic". Essential for protection against external pathogens and intrinsic degenerative processes, the inflammatory response becomes dysregulated with aging, leading to a persistent state of low-grade inflammation known as inflamm-aging. Of interest, inflammation has been recently recognized as a key factor in the pathogenesis of CV diseases, suggesting inflamm-aging as a possible driver of age-related CV afflictions and a plausible therapeutic target in this context. This review discusses the molecular pathways underlying inflamm-aging and their involvement in CV disease. Moreover, the potential of several anti-inflammatory approaches in this context is also reviewed

Inflammation and cardiovascular diseases: lessons from seminal clinical trials.

Abstract Inflammation has been long regarded as a key contributor to atherosclerosis. Inflammatory cells and soluble mediators play critical roles throughout arterial plaque development and accordingly, targeting inflammatory pathways effectively reduces atherosclerotic burden in animal models of cardiovascular (CV) diseases. Yet, clinical translation often led to inconclusive or even contradictory results. The Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS) followed by the Colchicine Cardiovascular Outcomes Trial (COLCOT) were the first two randomized clinical trials to convincingly demonstrate the effectiveness of specific anti-inflammatory treatments in the field of CV prevention, while other phase III trials—including the Cardiovascular Inflammation Reduction Trial one using methotrexate—were futile. This manuscript reviews the main characteristics and findings of recent anti-inflammatory Phase III trials in cardiology and discusses their similarities and differences in order to get further insights into the contribution of specific inflammatory pathways on CV outcomes. CANTOS and COLCOT demonstrated efficacy of two anti-inflammatory drugs (canakinumab and colchicine, respectively) in the secondary prevention of major adverse CV events (MACE) thus providing the first confirmation of the involvement of a specific inflammatory pathway in human atherosclerotic CV disease (ASCVD). Also, they highlighted the NOD-, LRR-, and pyrin domain-containing protein 3 inflammasome-related pathway as an effective therapeutic target to blunt ASCVD. In contrast, other trials interfering with a number of inflammasome-independent pathways failed to provide benefit. Lastly, all anti-inflammatory trials underscored the importance of balancing the risk of impaired host defence with an increase in infections and the prevention of MACE in CV patients with residual inflammatory risk

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)-α-induced as well as basal TF expression (P = 0.029, 0.0003, and 0.003 at maximal concentration), and TNF-α-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 NH2-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 consumptio

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 µg/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-α-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-α-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 DE