The role of TRPC6 in megakaryocytes and platelets

Blutplättchen sind kernlose Zellen, die eine zentrale Rolle bei der Thrombusbildung und dem Verschluss von Gefäßverletzungen spielen. Sie entstehen aus Vorläuferzellen, den sogenannten Megakaryozyten (MKs), die sich im Knochenmark finden. Für die Aktivierung der Blutplättchen ist die Konzentrationszunahme von intrazellulärem Calcium (Ca2+) von zentraler Bedeutung. Heute weiß man, dass in der Plättchenmembran mehrere Ca2+ Kanäle existieren, die am Calcium-Transport in die Zelle beteiligt sind. Einer dieser Kanäle, der kanonische transiente Rezeptor Kanal 6 (TRPC6), ist sowohl auf humanen als murinen Plättchen in hoher Konzentration exprimiert und konnte auch auf Megakaryozyten nachgewiesen werden. Wir haben in dieser Studie die Rolle des TRPC6 für die Entwicklung von MKs und für die Plättchenphysiologie untersucht. Zu diesem Zwecke generierten wir humane MKs aus humanen CD34+ hämatopoietischen Vorläuferzellen, die wir aus Nabelschnurblut isolierten. Die Zellen wurden 7-10 Tage in einem Zytokin-Cocktail kultiviert. Danach fanden sich große Zellen mit basophilem Zytoplasma in der Kultur, die die plättchenspezifischen Marker CD61, CD41 und CD42b auf der Oberfläche exprimierten. Der Nachweis dieser Marker zeigte an, dass die hämatopoietischen Stammzellen unter den gewählten Bedingungen zu MKs differenziert waren. Die generierten MKs enthielten hohe Konzentrationen von TRPC6 mRNA und Protein. Der Zusatz von Flufenamid-Säure, einem TRPC6 Aktivator, während der Kultivierung führte zu einem erhöhten Ca2+ Influx. Durch die Behandlung mit SKF96365, einem Inhibitor von TRPC Kanälen, wurde die Ca2+ Aufnahme blockiert und es kam zu einer Inhibition der MK- Proliferation. Wir schließen daraus, dass TRPC Kanäle bzw. TRPC6 für die Ca2+ Aufnahme während des MK Wachstums erforderlich sind. Um unsere in der Zellkultur gemachten Beobachtungen betreffend TRPC6 zu bestätigen untersuchten wir Blutplättchen und ihre Funktion in einem TRPC6 knock-out Mausmodell. Wir fanden, dass eine Behandlung mit Diacylglycerol (DAG), was bekanntermaßen die Calcium-Aufnahme mediiert, bei Plättchen von Wildtyp-Mäusen zu einer Zunahme von intrazellulärem Ca2+ führt, wohingegen bei TRPC6 -/- Plättchen kein Ca2+ Anstieg zu beobachten war. Interessanterweise unterschieden sich aber weder die Plättchengröße, noch die Plättchenzahl, die P-selektin Konzentration, die Integrin-Aktivierung, die Plättchenaggregation, die in vivo Thrombusbildung oder die Blutungszeit bei den TRPC6 -/- Mäusen von den Wildtyp Mäusen. Daraus könnte vi man schließen, dass bei Mäusen die über TRPC6 mediierte, DAG induzierte Ca2+ Aufnahme für die Plättchenphysiologie bzw. ihre Funktion nicht zwingend nötig ist. Allerdings ist nicht ausgeschlossen, dass TRPC6 Kanäle im Humansystem unter physiologischen Bedingungen, bei denen die Konzentration und Wirkung der Agonisten sehr präzise balanziert sind, eine Bedeutung für die Thrombusbildung haben.submitted by Gajalakshmi RamanathanAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Med. Univ., Diss., 2012OeBB(VLID)171582

The Microenvironment in Myeloproliferative Neoplasms

Chronic inflammation is a hallmark of myeloproliferative neoplasms (MPNs), with elevated levels of proinflammatory cytokines being commonly found in all 3 subtypes. Systemic inflammation is responsible for the constitutional symptoms, thrombosis risk, premature atherosclerosis, and disease evolution in MPN. Although the neoplastic clone and their differentiated progeny drive the inflammatory process, they also induce ancillary cytokine secretion from nonmalignant cells. Here, the authors describe the inflammatory milieu in MPN based on soluble factors and cellular mediators. They also discuss the prognostic value of cytokine measurements in patients with MPN and potential therapeutic strategies that target the cellular players in inflammation

Impact of Host, Lifestyle and Environmental Factors in the Pathogenesis of MPN.

Philadelphia-negative myeloproliferative neoplasms (MPNs) occur when there is over-production of myeloid cells stemming from hematopoietic stem cells with constitutive activation of JAK/STAT signaling, with JAK2V617F being the most commonly occurring somatic driver mutation. Chronic inflammation is a hallmark feature of MPNs and it is now evident that inflammation is not only a symptom of MPN but can also provoke development and precipitate progression of disease. Herein we have considered major MPN driver mutation independent host, lifestyle, and environmental factors in the pathogenesis of MPN based upon epidemiological and experimental data. In addition to the traditional risk factors such as advanced age, there is evidence to indicate that inflammatory stimuli such as smoking can promote and drive MPN clone emergence and expansion. Diet induced inflammation could also play a role in MPN clonal expansion. Recognition of factors associated with MPN development support lifestyle modifications as an emerging therapeutic tool to restrain inflammation and diminish MPN progression

Impact of Host, Lifestyle and Environmental Factors in the Pathogenesis of MPN.

Philadelphia-negative myeloproliferative neoplasms (MPNs) occur when there is over-production of myeloid cells stemming from hematopoietic stem cells with constitutive activation of JAK/STAT signaling, with JAK2V617F being the most commonly occurring somatic driver mutation. Chronic inflammation is a hallmark feature of MPNs and it is now evident that inflammation is not only a symptom of MPN but can also provoke development and precipitate progression of disease. Herein we have considered major MPN driver mutation independent host, lifestyle, and environmental factors in the pathogenesis of MPN based upon epidemiological and experimental data. In addition to the traditional risk factors such as advanced age, there is evidence to indicate that inflammatory stimuli such as smoking can promote and drive MPN clone emergence and expansion. Diet induced inflammation could also play a role in MPN clonal expansion. Recognition of factors associated with MPN development support lifestyle modifications as an emerging therapeutic tool to restrain inflammation and diminish MPN progression

Key Role of Inflammation in Myeloproliferative Neoplasms: Instigator of Disease Initiation, Progression. and Symptoms

Purpose of reviewChronic inflammation is a characteristic feature of myeloproliferative neoplasm (MPN) and impacts many aspects of the disease including initiation, progression, and symptomatology.Recent findingsThe chronic inflammatory state of MPN results from disruption of immune signaling pathways leading to overproduction of inflammatory cytokines by both the neoplastic clones and bystander immune cells. This chronic inflammation may allow for the neoplastic clone to gain a selective advantage. The symptomatic burden felt by MPN patients may be a result of the chronic inflammation associated with MPN, as several cytokines have been linked with different symptoms. Pharmacologic as well as nonpharmacologic treatments of the inflammatory component of this disease may lead to decreased symptomatic burden, prevention of disease progression, and improvement in overall disease trajectory. Inflammation plays a key role in the pathogenesis of MPN and represents an important therapeutic target

Cigarette smoking is associated with dose-dependent adverse effects on paraoxonase activity and fibrinogen in young women.

ContextSmoking is associated with increased fibrinogen and decreased paraoxonase (PON) activity, markers of inflammation and oxidative stress, in patients with coronary artery disease.ObjectiveWe tested the hypothesis that the adverse effect of smoking on these biomarkers of inflammation and oxidative stress would be detectable in otherwise healthy young female habitual smokers.Materials and methodsThirty-eight young women participated in the study (n = 20 habitual smokers, n = 18 non-smokers). Fibrinogen, PON-1 activity and HDL oxidant index (HOI) were measured.ResultsMean values of fibrinogen, PON-1 activity and log HOI were not different between the groups. Importantly, however, decreased PON-1 activity (rs = -0.51, p = 0.03) and increased fibrinogen (rs = 0.49, p = 0.04) were significantly correlated with increasing number of cigarettes smoked per day in habitual smokers.Discussion and conclusionCigarette smoking is associated with a dose-dependent adverse effect on PON-1 activity and fibrinogen in young women, which may have implications for future cardiovascular risk

N-acetylcysteine inhibits thrombosis in a murine model of myeloproliferative neoplasm

Thrombosis is a major cause of mortality in patients with myeloproliferative neoplasms (MPNs), though there is currently little to offer patients with MPN beyond aspirin and cytoreductive therapies such as hydroxyurea for primary prevention. Thrombogenesis in MPN involves multiple cellular mechanisms, including platelet activation and neutrophil-extracellular trap formation; therefore, an antithrombotic agent that targets one or more of these processes would be of therapeutic benefit in MPN. Here, we treated the JAK2V617F knockin mouse model of polycythemia vera with N-acetylcysteine (NAC), a sulfhydryl-containing compound with broad effects on glutathione replenishment, free radical scavenging, and reducing disulfide bonds, to investigate its antithrombotic effects in the context of MPN. Strikingly, NAC treatment extended the lifespan of JAK2V617F mice without impacting blood counts or splenomegaly. Using an acute pulmonary thrombosis model in vivo, we found that NAC reduced thrombus formation to a similar extent as the irreversible platelet inhibitor aspirin. In vitro analysis of platelet activation revealed that NAC reduced thrombin-induced platelet-leukocyte aggregate formation in JAK2V617F mice. Furthermore, NAC reduced neutrophil extracellular trap formation in primary human neutrophils from patients with MPN as well as healthy controls. These results provide evidence that N-acetylcysteine inhibits thrombosis in JAK2V617F mice and provide a pre-clinical rationale for investigating NAC as a therapeutic to reduce thrombotic risk in MPN