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

Myocardial infarction with non-obstructive coronary arteries: what is the prognosis?

Myocardial infarction in the absence of obstructive coronary stenosis (MINOCA) is a syndrome with several causes, characterized by clinical evidence of myocardial infarction and coronary angiographically normal or almost normal (stenosis <= 50%). MINOCAs represent about 10% of acute coronary syndromes. The causes of MINOCA are manifold and can be classified on the basis of the mechanism in epicardial (unstable plaque not manifested by angiography, epicardial spasm and coronary dissection) or microvascular. The latter in turn can be divided into intrinsic (microvascular spasm, Takotsubo syndrome and coronary embolization) and extrinsic (myocarditis). In the former, the dysfunctional microcirculation causes myocardial necrosis due to reduction of the lumen due to vasoconstriction and / or obstruction, while in the latter, the compression of the lumen occurs ab extrinsic due to myocardial edema. Note that the prognosis of MINOCA is extremely variable and depends on the underlying cause with high risk clinical subsets. A correct diagnostic procedure includes first level tests (clinical / anamnestic examination, ECG, myocardial necrosis enzyme dosage, trans-thoracic echocardiogram, coronary angiography, ventriculogram) and second level tests (intracoronary imaging, coronary vasomotor test, cardiac nuclear magnetic resonance and trans-esophageal or contrast ultrasound). Through this process, it is possible to identify the cause of MINOCA, fundamental for targeting therapy on the disease mechanism, thus constituting a typical example of precision medicine

k-Factorization and Small-x Anomalous Dimensions

We investigate the consistency requirements of the next-to leading BFKL equation with the renormalization group, with particular emphasis on running coupling effects and NL anomalous dimensions. We show that, despite some model dependence of the bare hard Pomeron, such consistency holds at leading twist level, provided the effective variable $\alpha_s(t) log(1/x)$ is not too large. We give a unified view of resummation formulas for coefficient functions and anomalous dimensions in the Q_0-scheme and we discuss in detail the new one for the $q\bar{q}$ contributions to the gluon channel.Comment: Latex2e, 44 pages including 7 PostScript figure

Heavy quark production as sensitive test for an improved description of high energy hadron collisions

QCD dynamics at small quark and gluon momentum fractions or large total energy, which plays a major role for HERA, the Tevatron, RHIC and LHC physics, is still poorly understood. For one of the simplest processes, namely bottom-antibottom production, next-to-leading-order perturbation theory fails. We show that the combination of two recently developed theoretical concepts, the k_perp-factorization and the next-to-leading-logarithmic-approximation BFKL vertex, gives perfect agreement with data. One can therefore hope that these concepts provide a valuable foundation for the description of other high energy processes.Comment: RevTeX, 4 pages, 7 figures titel and abstract changed, several formulations modified in the text, 1 figure droppe

Irreducible part of the next-to-leading BFKL kernel

On the basis of previous work by Fadin, Lipatov, and collaborators, and of our group, we extract the "irreducible" part of the next-to-leading (NL) BFKL kernel, we compute its (IR finite) eigenvalue function, and we discuss its implications for small-x structure functions. We find consistent running coupling effects and sizable NL corrections to the Pomeron intercept and to the gluon anomalous dimension. The qualitative effect of such corrections is to smooth out the small-x rise of structure functions at low values of Q2. A more quantitative analysis will be possible after the extraction of some additional, energy-scale dependent contributions to the kernel, which are not treated here.Comment: 16 pages, LaTex2e, including 3 eps figure

Resumming double logarithms in the QCD evolution of color dipoles

The higher-order perturbative corrections, beyond leading logarithmic accuracy, to the BFKL evolution in QCD at high energy are well known to suffer from a severe lack-of-convergence problem, due to radiative corrections enhanced by double collinear logarithms. Via an explicit calculation of Feynman graphs in light cone (time-ordered) perturbation theory, we show that the corrections enhanced by double logarithms (either energy-collinear, or double collinear) are associated with soft gluon emissions which are strictly ordered in lifetime. These corrections can be resummed to all orders by solving an evolution equation which is non-local in rapidity. This equation can be equivalently rewritten in local form, but with modified kernel and initial conditions, which resum double collinear logs to all orders. We extend this resummation to the next-to-leading order BFKL and BK equations. The first numerical studies of the collinearly-improved BK equation demonstrate the essential role of the resummation in both stabilizing and slowing down the evolution.Comment: 16 pages, 5 figure

Expanding running coupling effects in the hard Pomeron

We study QCD hard processes at scales of order k^2 > Lambda^2 in the limit in which the beta-function coefficient - b is taken to be small, but alphas(k) is kept fixed. The (nonperturbative) Pomeron is exponentially suppressed in this limit, making it possible to define purely perturbative high-energy Green's functions. The hard Pomeron exponent acquires diffusion and running coupling corrections which can be expanded in the b parameter and turn out to be dependent on the effective coupling b alphas^2 Y. We provide a general setup for this b-expansion and we calculate the first few terms both analytically and numerically.Comment: 36 pages, 15 figures, additional references adde