Defining murine organogenesis at single-cell resolution reveals a role for the leukotriene pathway in regulating blood progenitor formation.

During gastrulation, cell types from all three germ layers are specified and the basic body plan is established 1 . However, molecular analysis of this key developmental stage has been hampered by limited cell numbers and a paucity of markers. Single-cell RNA sequencing circumvents these problems, but has so far been limited to specific organ systems 2 . Here, we report single-cell transcriptomic characterization of >20,000 cells immediately following gastrulation at E8.25 of mouse development. We identify 20 major cell types, which frequently contain substructure, including three distinct signatures in early foregut cells. Pseudo-space ordering of somitic progenitor cells identifies dynamic waves of transcription and candidate regulators, which are validated by molecular characterization of spatially resolved regions of the embryo. Within the endothelial population, cells that transition from haemogenic endothelial to erythro-myeloid progenitors specifically express Alox5 and its co-factor Alox5ap, which control leukotriene production. Functional assays using mouse embryonic stem cells demonstrate that leukotrienes promote haematopoietic progenitor cell generation. Thus, this comprehensive single-cell map can be exploited to reveal previously unrecognized pathways that contribute to tissue development

Effect of wood smoke exposure on vascular function and thrombus formation in healthy fire fighters

Background: Myocardial infarction is the leading cause of death in fire fighters and has been linked with exposure to air pollution and fire suppression duties. We therefore investigated the effects of wood smoke exposure on vascular vasomotor and fibrinolytic function, and thrombus formation in healthy fire fighters. Methods: In a double-blind randomized cross-over study, 16 healthy male fire fighters were exposed to wood smoke (~1 mg/m3 particulate matter concentration) or filtered air for one hour during intermittent exercise. Arterial pressure and stiffness were measured before and immediately after exposure, and forearm blood flow was measured during intra-brachial infusion of endothelium-dependent and -independent vasodilators 4–6 hours after exposure. Thrombus formation was assessed using the ex vivo Badimon chamber at 2 hours, and platelet activation was measured using flow cytometry for up to 24 hours after the exposure. Results: Compared to filtered air, exposure to wood smoke increased blood carboxyhaemoglobin concentrations (1.3% versus 0.8%; P &lt; 0.001), but had no effect on arterial pressure, augmentation index or pulse wave velocity (P &gt; 0.05 for all). Whilst there was a dose-dependent increase in forearm blood flow with each vasodilator (P &lt; 0.01 for all), there were no differences in blood flow responses to acetylcholine, sodium nitroprusside or verapamil between exposures (P &gt; 0.05 for all). Following exposure to wood smoke, vasodilatation to bradykinin increased (P = 0.003), but there was no effect on bradykinin-induced tissue-plasminogen activator release, thrombus area or markers of platelet activation (P &gt; 0.05 for all). Conclusions: Wood smoke exposure does not impair vascular vasomotor or fibrinolytic function, or increase thrombus formation in fire fighters. Acute cardiovascular events following fire suppression may be precipitated by exposure to other air pollutants or through other mechanisms, such as strenuous physical exertion and dehydration.Originally included in thesis in manuscript form.</p

Blood platelet behaviour and platelet-von Willebrand Factor interaction under force

Platelet integrity and dynamics are crucial in hemostasis and thrombosis. During these processes, circulating disc shaped platelets are recruited from the blood stream to sites of vascular injuries. The initial platelet and plasma protein, von Willebrand factor (vWF), interaction occurs platelet activation independent. Depending on the combination of laminar blood flow shear stress and the platelet-vWF interplay, the unactivated platelets either adhere or form tethers by pulling membrane pieces from the moving platelets. The irreversible attachment step of activated platelets to vWF induces spreading and in contrast, to collagen, which is exposed after endothelial injury, aggregation. Aim of this interdisciplinary thesis was to bridge the gap between in vitro measurements and the understanding of factors influencing the platelet-vWF interaction. Specific focus was given on individual phenomena as well as collective phenomena. The single investigations involved variations in the mechanical properties of the platelets as well as in the vWF. To explore the role of cortical tension of the platelet envelope, their cytoskeleton was controlled disrupted. Variations in the cholesterol concentration, which is a component of the plasma membrane, were quantified to measure the impact on the investigated platelet functions as platelet adhesion and tether formation. Altering the size distribution of the vWF multimer and mutations in the vWF protein were investigated to clarify the role of the vWF functionality on the platelet-vWF interplay. The formation of platelet-vWF aggregates was characterised to study the conditions of stenosis and atherosclerotic occlusions. The multi step experiments discuss variations in the hematocrit (HCT), especially the impact of platelet-red blood cell collisions and the flattening of the flow profile by higher HCTs. The platelet-tumor cells interactions were studied to clarify the role of tumor cells in platelet activation, aggregation and receptor transfer.Die Plättchenintegrität und deren dynamisches Verhalten sind entscheidende Faktoren in der Hämostase und Thrombose. Während dieser wichtigen Prozesse werden scheibenförmige Plättchen vom Blutfluss rekrutiert und an verletzte Gefäße geführt. Die initiale Wechselwirkung von Plättchen mit einem Plasmaprotein von Willebrand Faktor (vWF), findet ohne vorherige Plättchenaktivierung statt. Abhängig von der Kombination aus laminarem Blutfluss und der Plättchen-vWF Interaktion, adherieren die Plättchen oder formen Tether durch das Ziehen von Membranstücken aus den sich bewegenden Plättchen. Die irreversible Adhäsion induziert das sogenannte Spreading, bei welchem die aktivierten Plättchen an den vWF anhaften. Im Gegensatz dazu nennt man die Adhäsion der Plättchen auf Kollagen Aggregation. Kollagen wird nach Endothelverletzungen exponiert. Das Ziel der vorliegenden Arbeit war es, eine Brücke zwischen in vitro Messungen und den Einflussfaktoren der Plättchen-vWF Wechselwirkung zu spannen. Spezieller Fokus wurde einerseits auf Einzelphänomene als auch auf kooperative Phänomene gelegt. Zu den Einzeluntersuchen zählten Veränderungen der mechanischen Eigenschaften des Plättchens sowie im vWF. Um die Rolle der kortikalen Spannung der Plättchenhülle zu untersuchen, wurde das Plättchenzytoskelett kontrolliert zerstört. Veränderungen in der Cholesterinkonzentration wurden quantifiziert, um den Einfluss auf das untersuchte Plättchen- und Tetherformationsverhalten herauszustellen. Veränderungen in der vWF Multimerverteilung und Mutationen des vWFs wurden untersucht, um die Auswirkungen der vWF Funktionalität auf das Plättchen-vWF Wechselspiel darzulegen. Mit Hilfe der Formation von rollenden Plättchen-vWF Aggregaten wurde der Einfluss von Stenosen und atheriosklerotischen Verengungen untersucht. Die Mehrkomponentenuntersuchungen behandelten Veränderungen im Hämatokrit (HCT) vor allem den Einfluss der Plättchen-Erythrozyten Kollisionen und das Abflachen des Flussprofils. Zudem wurde die Plättchen-Tumorzellen Wechselwirkung untersucht, um die Auswirkung der Tumorzellen auf die Plättchenaktivierung, deren Aggregation, und der Rezeptorübertragung aufzuzeigen

Hematocrit and flow rate regulate the adhesion of platelets to von Willebrand factor

Primary hemostasis and blood clotting is known to be influenced by the red blood cell volume fraction (hematocrit) in blood. Depressed or elevated levels of red blood cells can lead to vascular perfusion problems ranging from bleeding to thrombus formation. The early stage of hemostasis and thus blood clotting in all vessel sections from the arterial to the venous system involves the adhesion of platelets to von Willebrand factor. Here we present experimental and theoretical results showing that the adhesion probability of platelets to von Willebrand factor is strongly and nonlinearly dependent on hematocrit and flow rate. Interestingly, the actual binding forces are not markedly different, which suggest that the origin of such behavior is in the distribution of platelets. Using hydrodynamic simulations of a simple model, we explicitly show that the higher the hematocrit and the flow rate, the larger the amount of platelets residing close to the wall. Our simulation results, which are in excellent agreement with the experimental observations, explain why such phenomena occur. We believe that the nonhomogeneous red blood cell distribution as well as the shear dependent hydrodynamic interaction is key for the accumulation of platelets on the vessel wall. The work we present here is an important step forward from our earlier work on single molecules and extends into the collective cellular behavior of whole blood. It sheds new light on the correlation between hematocrit and the initial steps in hemostasis and thrombosis, and outlines advances for the treatment of vascular diseases associated with high levels of red blood cells. These results are not only highly relevant for the field of hemostasis and the physics of blood clotting but are also of powerful impact in applied science most obviously in drug delivery and colloidal science.National Science Foundation (U.S.) (CAREER Award 1054671