Filtern
Volltext vorhanden
- ja (91)
Gehört zur Bibliographie
- ja (91)
Erscheinungsjahr
Dokumenttyp
Schlagworte
- platelets (11)
- platelet (10)
- LASP1 (7)
- ischemic stroke (7)
- atherosclerosis (6)
- Arteriosklerose (5)
- Thrombozyt (5)
- megakaryocytes (5)
- thrombo-inflammation (5)
- Atherosklerose (4)
- CXCR4 (4)
- glycoprotein VI (4)
- platelet activation (4)
- thrombosis (4)
- Atherosclerosis (3)
- Cytoskeleton (3)
- Megakaryozyt (3)
- cytoskeleton (3)
- cytotoxic T cells (3)
- epigenetics (3)
- inflammation (3)
- mouse (3)
- platelet aggregation (3)
- thrombin (3)
- thrombopoiesis (3)
- AAA (2)
- CML (2)
- COVID-19 (2)
- Caenorhabditis elegans (2)
- Herzinfarkt (2)
- Knochenmark (2)
- Makrophage (2)
- Phagozytose (2)
- Zellskelett (2)
- Zelltod (2)
- actin cytoskeleton (2)
- aortic arch (2)
- blood (2)
- bone marrow (2)
- breast cancer (2)
- collagens (2)
- glycoprotein receptor Ib (2)
- hematology (2)
- ischemic penumbra (2)
- matrix metalloproteinases (2)
- mice (2)
- middle cerebral artery occlusion (2)
- mouse models (2)
- platelet receptors (2)
- pulse wave velocity (2)
- signaling (2)
- stem cells (2)
- transcriptional regulation (2)
- translational research (2)
- wall shear stress (2)
- 3D image analysis (1)
- 4D flow (1)
- 4D flow MRI (1)
- 7-Dehydrocholesterol (1)
- A20 (1)
- ADAM10 (1)
- AKT (1)
- AKT1 (1)
- AP-1 (1)
- AP1 (1)
- Abszision (1)
- Actin (1)
- Actin-bindende Proteine (1)
- Adhesion and degranulation promoting adapter protein (1)
- Akt (1)
- Artery Models (1)
- Atherogenese (1)
- Autophagie (1)
- B cell (1)
- B cell maturation (1)
- B cell receptor (1)
- BCR‐ABL (1)
- Batf3 (1)
- Bericht (1)
- Biofabrication (1)
- Biomarker (1)
- CCR6 (1)
- CD147 (1)
- CD2AP (1)
- CD8+ T-Zellen (1)
- CD8\(^+\) T cells (1)
- CMR (1)
- CVD (1)
- Canis lupus familiaris (1)
- Cdc42 (1)
- Chemokine (1)
- Cholesterin (1)
- Cholesterol (1)
- Chronisch-myeloische Leukämie (1)
- Collaborative Research Center (1)
- Cyclophilin A (1)
- DHCR7 (1)
- Dendritische Zelle (1)
- Differential RNA-sequencing (1)
- Domäne <Biochemie> (1)
- Dünndarm (1)
- E2 conjugating enzyme (1)
- E3 ligating enzyme (1)
- EMMPRIN (1)
- ERK map kinease (1)
- ERK1/2 (1)
- Epigenetik (1)
- Experimental Biomedicine (1)
- Extracellular matrix proteins (1)
- Extrazelluläre Matrix (1)
- Ferroptose (1)
- Ferroptosis (1)
- GABAA receptors (1)
- GABP (1)
- GPR54 (1)
- GPVI (1)
- GSK-3β (1)
- Ganzkörperbestrahlung (1)
- Graft-versus-host disease (GvHD) (1)
- HIF-1α (1)
- Helicobacter pylori (1)
- Hemodynamics (1)
- Histologie (1)
- Hypoxie-induzierbarer Faktor (1)
- Hämodynamik (1)
- IgD (1)
- IgM (1)
- Immunität <Medizin> (1)
- JAQ1 (1)
- KAT/HAT (1)
- KISS1 receptor (1)
- KISS1-54 (1)
- Kardiovaskuläre Erkrankungen (1)
- Knochenmarktransplantation (1)
- LASP2 (1)
- LC3-associated phagocytosis (1)
- LC3-assoziierte Phagozytose (1)
- MGL (1)
- MRI (1)
- Macrophages (1)
- Mast cells (1)
- Megakaryocyte (1)
- Megakaryocytes (1)
- Monozyten (1)
- Myocardial infarction (1)
- Neutrophils (1)
- Optogenetik (1)
- Orai2 (1)
- PD-L1 (1)
- PET (1)
- PWV (1)
- Platelet (1)
- Polkörper (1)
- Proteininteraktion (1)
- Proteinsynthese (1)
- RKIP (1)
- Rekrutierung (1)
- RhoA (1)
- RhoF (1)
- SARS-CoV-2 (1)
- SET7 (1)
- Sepsis (1)
- Septine (1)
- Single-cell RNA Sequencing (1)
- Snail1 stability (1)
- Sonderforschungsbereich Transregio 240 (1)
- Strumpellin (1)
- Syk (1)
- T cell (1)
- T-Lymphozyt (1)
- T-Lymphozyten (1)
- T-cell epitope (1)
- T-cells (1)
- Tet‐inducible system (1)
- Thrombopoiesis (1)
- Thrombozytenfunktion (1)
- Thrombozytopenie (1)
- Tissue Engineering (1)
- TspanC8 (1)
- Urothelkrebs (1)
- WASH complex (1)
- WSS (1)
- ZF1 degradation assay (1)
- ZIP (1)
- Zellteilung (1)
- [\(^{68}\)]KISS1-54 (1)
- acetylation (1)
- acetyltransferases (1)
- actin-binding proteins (1)
- actins (1)
- acute Respiratory Distress Syndrome (1)
- adaptor protein Swiprosin-1/EFhd2 (1)
- adipose tissue (1)
- algorithm (1)
- alpha-IIb beta-3 (1)
- angiotensin (1)
- animal models (1)
- arterial elasticity (1)
- arterial thrombus formation (1)
- basic (laboratory) research / science (1)
- bioenergetics (1)
- bioinformatics (1)
- bleding disorders other than hemophilia (1)
- bleeding (1)
- blood coagulation (1)
- blood flow (1)
- blood platelets (1)
- body weight (1)
- brain (1)
- c-Fos (1)
- cag pathogenicity island (1)
- campylobacter jejuni infection (1)
- cancer (1)
- cardiovascular MRI (1)
- cardiovascular disease (1)
- carotid arteries (1)
- cascade (1)
- cerebellum (1)
- cerebrovascular disorders (1)
- checkpoint inhibitors (1)
- chemokines (1)
- coagulation (1)
- coagulation and hemostasis (1)
- coagulation system (1)
- coatings (1)
- coffin-lowry-syndrome (1)
- collagen (1)
- complex (1)
- controllability (1)
- cyclase-associated protein (1)
- cyclase-associated protein 2 (1)
- cytoskeletal proteins (1)
- deficiency (1)
- dendritic cells (1)
- dendritic growth (1)
- dentate gyrus (1)
- disaggregation (1)
- disease (1)
- dog (1)
- drug repurposing (1)
- early diagnosis (1)
- ectopic release (1)
- embryos (1)
- en bloc transfer (1)
- endosomal trafficking (1)
- endothelial cell (1)
- endothelial cells (1)
- enzyme regulation (1)
- epithelial cells (1)
- epitope prediction (1)
- evolution (1)
- extracellular matrix (1)
- factor XII (1)
- fibrin (1)
- filopodia (1)
- flow (1)
- flow dynamics (1)
- fluorescence microscopy (1)
- fostamatinib (1)
- genes (1)
- genetic modification (1)
- gephyrin (1)
- glaucoma (1)
- glycine receptors (1)
- glycoprotein Ib (1)
- glycoprotein Ibα (1)
- glycoprotein receptor Ibα (1)
- graft survival (1)
- granule cells (1)
- guidelines (1)
- haemostasis (1)
- heart (1)
- hemorrhage (1)
- hemostasis and thrombosis (1)
- hemostasis, (1)
- hepatology (1)
- hippocampus (1)
- histologic diversity (1)
- histology (1)
- histone acetylation (1)
- homeostasisIon channels (1)
- human (1)
- human hematopoiesis (1)
- human tumor cell lines (1)
- hypothalamus (1)
- iPS cells (1)
- imaging the immune system (1)
- imiquimod (1)
- immune-informatics (1)
- immunoglobulin repertoire (1)
- immunoglobulin superfamily (1)
- immunotherapy (1)
- impedance aggregometry; WHOLE-BLOOD THROMBOELASTOMETRY; DEFINITION; DISEASE (1)
- in vitro and in vivo thrombus formation (1)
- infection (1)
- influenza (1)
- inherited macrothrombocytopenia (1)
- inhibitory post-synaptic specialization (1)
- insulin (1)
- insulin resistance (1)
- integrin α2 (1)
- integrins (1)
- interaction (1)
- interactome (1)
- interspecies comparison (1)
- intracranial bleeding (1)
- islet transplantation (1)
- kisspeptin (1)
- laboratory animals (1)
- large animal models (1)
- leukemia (1)
- light sheet fluorescence microscopy (1)
- lncRNAs (1)
- long-term potentation (1)
- lung cancer (1)
- lung injury (1)
- lymphocyte differentiation (1)
- magnetic resonance imaging (1)
- mapping (1)
- megakaryocyte (1)
- metalloproteinase (1)
- metals (1)
- miRNA expression (1)
- miRNAs (1)
- microfluidics (1)
- microscopy (1)
- midbody remnant (1)
- molecular biology (1)
- monocyte (1)
- monocyte-platelet aggregates (1)
- moonlighting protein (1)
- mutation (1)
- myeloablation (1)
- nephrocyte (1)
- network (1)
- neurite outgrowth (1)
- neurology (1)
- neuronal dendrites (1)
- neuronal differentiation (1)
- neurons (1)
- neurotrophic factor (1)
- neurotrophic factors (1)
- neutrophil (1)
- nilotinib (1)
- non-invasive biomarkers (1)
- nuclear role (1)
- nucleus (1)
- occlusion (1)
- optical clearing (1)
- optogenetics (1)
- organoid (1)
- pathway analysis (1)
- phagosome maturation (1)
- phosphatidic acid (1)
- phospholipase D (1)
- phosphorylation (1)
- pig (1)
- pigmentation (1)
- plaque (1)
- plaque characteristics (1)
- platelet biogenesis (1)
- platelet degranulation (1)
- platelet disorders (1)
- platelet inhibition (1)
- platelet-neutrophil complexes (PNCs) (1)
- pneumonia (1)
- point of care testing (1)
- polymers (1)
- popliteal aneurysm (1)
- positive selection (1)
- positron emission tomography (1)
- precursor cells (1)
- proplatelets (1)
- protein RSK2 (1)
- proteome (1)
- psoriasis (1)
- quantification (1)
- radial (1)
- recombinant tissue-type plasminogen activator (1)
- recruitment (1)
- regulatory circuit downstream (1)
- research infrastructure (1)
- retroviral vectors (1)
- second messenger (1)
- self-navigation (1)
- sequential addition (1)
- serotonin (1)
- shedding (1)
- signaling network (1)
- single cell RNA sequencing (1)
- slit membrane (1)
- spatiotemporal thrombus (1)
- stent implantation (1)
- stroke (1)
- structure (1)
- tMCAO (1)
- tetraspanin (1)
- therapy (1)
- thrombocyte (1)
- thrombocytopenia (1)
- thromboelastometry (1)
- thrombus (1)
- transcriptome (1)
- transendothelial migration (1)
- transient middle cerebral artery (1)
- transient middle cerebral artery occlusion (1)
- tumor microenvironment (1)
- tyrosine kinase (1)
- ubiquitin (1)
- ultrahigh-field MRI (1)
- vascular structure (1)
- viral infection (1)
- zinc (1)
Institut
- Institut für Experimentelle Biomedizin (91) (entfernen)
Sonstige beteiligte Institutionen
Within this thesis, three main approaches for the assessment and investigation of altered hemodynamics like wall shear stress, oscillatory shear index and the arterial pulse wave velocity in atherosclerosis development and progression were conducted:
1. The establishment of a fast method for the simultaneous assessment of 3D WSS and PWV in the complete murine aortic arch via high-resolution 4D-flow MRI
2. The utilization of serial in vivo measurements in atherosclerotic mouse models using high-resolution 4D-flow MRI, which were divided into studies describing altered hemodynamics in late and early atherosclerosis
3. The development of tissue-engineered artery models for the controllable application and variation of hemodynamic and biologic parameters, divided in native artery models and biofabricated artery models, aiming for the investigation of the relationship between atherogenesis and hemodynamics
Chapter 2 describes the establishment of a method for the simultaneous measurement of 3D WSS and PWV in the murine aortic arch at, using ultra high-field MRI at 17.6T [16], based on the previously published method for fast, self-navigated wall shear stress measurements in the murine aortic arch using radial 4D-phase contrast MRI at 17.6 T [4]. This work is based on the collective work of Dr. Patrick Winter, who developed the method and the author of this thesis, Kristina Andelovic, who performed the experiments and statistical analyses. As the method described in this chapter is basis for the following in vivo studies and undividable into the sub-parts of the contributors without losing important information, this chapter was not split into the single parts to provide fundamental information about the measurement and analysis methods and therefore better understandability for the following studies. The main challenge in this chapter was to overcome the issue of the need for a high spatial resolution to determine the velocity gradients at the vascular wall for the WSS quantification and a high temporal resolution for the assessment of the PWV without prolonging the acquisition time due to the need for two separate measurements. Moreover, for a full coverage of the hemodynamics in the murine aortic arch, a 3D measurement is needed, which was achieved by utilization of retrospective navigation and radial trajectories, enabling a highly flexible reconstruction framework to either reconstruct images at lower spatial resolution and higher frame rates for the acquisition of the PWV or higher spatial resolution and lower frame rates for the acquisition of the 3D WSS in a reasonable measurement time of only 35 minutes. This enabled the in vivo assessment of all relevant hemodynamic parameters related to atherosclerosis development and progression in one experimental session. This method was validated in healthy wild type and atherosclerotic Apoe-/- mice, indicating no differences in robustness between pathological and healthy mice.
The heterogeneous distribution of plaque development and arterial stiffening in atherosclerosis [10, 12], however, points out the importance of local PWV measurements. Therefore, future studies should focus on the 3D acquisition of the local PWV in the murine aortic arch based on the presented method, in order to enable spatially resolved correlations of local arterial stiffness with other hemodynamic parameters and plaque composition.
In Chapter 3, the previously established methods were used for the investigation of changing aortic hemodynamics during ageing and atherosclerosis in healthy wild type and atherosclerotic Apoe-/- mice using the previously established methods [4, 16] based on high-resolution 4D-flow MRI. In this work, serial measurements of healthy and atherosclerotic mice were conducted to track all changes in hemodynamics in the complete aortic arch over time. Moreover, spatially resolved 2D projection maps of WSS and OSI of the complete aortic arch were generated. This important feature allowed for the pixel-wise statistical analysis of inter- and intragroup hemodynamic changes over time and most importantly – at a glance. The study revealed converse differences of local hemodynamic profiles in healthy WT and atherosclerotic Apoe−/− mice, with decreasing longWSS and increasing OSI, while showing constant PWV in healthy mice and increasing longWSS and decreasing OSI, while showing increased PWV in diseased mice. Moreover, spatially resolved correlations between WSS, PWV, plaque and vessel wall characteristics were enabled, giving detailed insights into coherences between hemodynamics and plaque composition. Here, the circWSS was identified as a potential marker of plaque size and composition in advanced atherosclerosis. Moreover, correlations with PWV values identified the maximum radStrain could serve as a potential marker for vascular elasticity. This study demonstrated the feasibility and utility of high-resolution 4D flow MRI to spatially resolve, visualize and analyze statistical differences in all relevant hemodynamic parameters over time and between healthy and diseased mice, which could significantly improve our understanding of plaque progression towards vulnerability. In future studies the relation of vascular elasticity and radial strain should be further investigated and validated with local PWV measurements and CFD.
Moreover, the 2D histological datasets were not reflecting the 3D properties and regional characteristics of the atherosclerotic plaques. Therefore, future studies will include 3D plaque volume and composition analysis like morphological measurements with MRI or light-sheet microscopy to further improve the analysis of the relationship between hemodynamics and atherosclerosis.
Chapter 4 aimed at the description and investigation of hemodynamics in early stages of atherosclerosis. Moreover, this study included measurements of hemodynamics at baseline levels in healthy WT and atherosclerotic mouse models. Due to the lack of hemodynamic-related studies in Ldlr-/- mice, which are the most used mouse models in atherosclerosis research together with the Apoe-/- mouse model, this model was included in this study to describe changing hemodynamics in the aortic arch at baseline levels and during early atherosclerosis development and progression for the first time. In this study, distinct differences in aortic geometries of these mouse models at baseline levels were described for the first time, which result in significantly different flow- and WSS profiles in the Ldlr-/- mouse model. Further basal characterization of different parameters revealed only characteristic differences in lipid profiles, proving that the geometry is highly influencing the local WSS in these models. Most interestingly, calculation of the atherogenic index of plasma revealed a significantly higher risk in Ldlr-/- mice with ongoing atherosclerosis development, but significantly greater plaque areas in the aortic arch of Apoe-/- mice. Due to the given basal WSS and OSI profile in these two mouse models – two parameters highly influencing plaque development and progression – there is evidence that the regional plaque development differs between these mouse models during very early atherogenesis.
Therefore, future studies should focus on the spatiotemporal evaluation of plaque development and composition in the three defined aortic regions using morphological measurements with MRI or 3D histological analyses like LSFM. Moreover, this study offers an excellent basis for future studies incorporating CFD simulations, analyzing the different measured parameter combinations (e.g., aortic geometry of the Ldlr-/- mouse with the lipid profile of the Apoe-/- mouse), simulating the resulting plaque development and composition. This could help to understand the complex interplay between altered hemodynamics, serum lipids and atherosclerosis and significantly improve our basic understanding of key factors initiating atherosclerosis development.
Chapter 5 describes the establishment of a tissue-engineered artery model, which is based on native, decellularized porcine carotid artery scaffolds, cultured in a MRI-suitable bioreactor-system [23] for the investigation of hemodynamic-related atherosclerosis development in a controllable manner, using the previously established methods for WSS and PWV assessment [4, 16]. This in vitro artery model aimed for the reduction of animal experiments, while simultaneously offering a simplified, but completely controllable physical and biological environment. For this, a very fast and gentle decellularization protocol was established in a first step, which resulted in porcine carotid artery scaffolds showing complete acellularity while maintaining the extracellular matrix composition, overall ultrastructure and mechanical strength of native arteries. Moreover, a good cellular adhesion and proliferation was achieved, which was evaluated with isolated human blood outgrowth endothelial cells. Most importantly, an MRI-suitable artery chamber was designed for the simultaneous cultivation and assessment of high-resolution 4D hemodynamics in the described artery models. Using high-resolution 4D-flow MRI, the bioreactor system was proven to be suitable to quantify the volume flow, the two components of the WSS and the radStrain as well as the PWV in artery models, with obtained values being comparable to values found in literature for in vivo measurements. Moreover, the identification of first atherosclerotic processes like intimal thickening is achievable by three-dimensional assessment of the vessel wall morphology in the in vitro models. However, one limitation is the lack of a medial smooth muscle cell layer due to the dense ECM. Here, the utilization of the laser-cutting technology for the generation of holes and / or pits on a microscale, eventually enabling seeding of the media with SMCs showed promising results in a first try and should be further investigated in future studies. Therefore, the proposed artery model possesses all relevant components for the extension to an atherosclerosis model which may pave the way towards a significant improvement of our understanding of the key mechanisms in atherogenesis.
Chapter 6 describes the development of an easy-to-prepare, low cost and fully customizable artery model based on biomaterials. Here, thermoresponsive sacrificial scaffolds, processed with the technique of MEW were used for the creation of variable, biomimetic shapes to mimic the geometric properties of the aortic arch, consisting of both, bifurcations and curvatures. After embedding the sacrificial scaffold into a gelatin-hydrogel containing SMCs, it was crosslinked with bacterial transglutaminase before dissolution and flushing of the sacrificial scaffold. The hereby generated channel was subsequently seeded with ECs, resulting in an easy-to-prepare, fast and low-cost artery model. In contrast to the native artery model, this model is therefore more variable in size and shape and offers the possibility to include smooth muscle cells from the beginning. Moreover, a custom-built and highly adaptable perfusion chamber was designed specifically for the scaffold structure, which enabled a one-step creation and simultaneously offering the possibility for dynamic cultivation of the artery models, making it an excellent basis for the development of in vitro disease test systems for e.g., flow-related atherosclerosis research. Due to time constraints, the extension to an atherosclerosis model could not be achieved within the scope of this thesis. Therefore, future studies will focus on the development and validation of an in vitro atherosclerosis model based on the proposed bi- and three-layered artery models.
In conclusion, this thesis paved the way for a fast acquisition and detailed analyses of changing hemodynamics during atherosclerosis development and progression, including spatially resolved analyses of all relevant hemodynamic parameters over time and in between different groups. Moreover, to reduce animal experiments, while gaining control over various parameters influencing atherosclerosis development, promising artery models were established, which have the potential to serve as a new platform for basic atherosclerosis research.
Besides their central role in haemostasis and thrombosis, platelets are increasingly recognised as versatile effector cells in inflammation, the innate and adaptive immune response, extracellular matrix reorganisation and fibrosis, maintenance of barrier and organ integrity, and host response to pathogens. These platelet functions, referred to as thrombo-inflammation and immunothrombosis, have gained major attention in the COVID-19 pandemic, where patients develop an inflammatory disease state with severe and life-threatening thromboembolic complications. In the CRC/TR 240, a highly interdisciplinary team of basic, translational and clinical scientists explored these emerging roles of platelets with the aim to develop novel treatment concepts for cardiovascular disorders and beyond. We have i) unravelled mechanisms leading to life-threatening thromboembolic complica-tions following vaccination against SARS-CoV-2 with adenoviral vector-based vaccines, ii) identified unrecognised functions of platelet receptors and their regulation, offering new potential targets for pharmacological intervention and iii) developed new methodology to study the biology of megakar-yocytes (MKs), the precursor cells of platelets in the bone marrow, which lay the foundation for the modulation of platelet biogenesis and function. The projects of the CRC/TR 240 built on the unique expertise of our research network and focussed on the following complementary fields: (A) Cell bi-ology of megakaryocytes and platelets and (B) Platelets as regulators and effectors in disease. To achieve this aim, we followed a comprehensive approach starting out from in vitro systems and animal models to clinical research with large prospective patient cohorts and data-/biobanking. Despite the comparably short funding period the CRC/TR 240 discovered basic new mechanisms of platelet biogenesis, signal transduction and effector function and identified potential MK/platelet-specific molecular targets for diagnosis and therapy of thrombotic, haemorrhagic and thrombo-inflammatory disease states.
Atherosklerose ist eine chronisch inflammatorische Erkrankung der Gefäßwände, bei der sowohl die angeborene als auch die erworbene Immunantwort beteiligt ist. Von Monozyten abstammende Makrophagen spielen eine Schlüsselrolle bei der Entstehung atherosklerotischer Läsionen. Durch die Aufnahme modifizierte Lipide (z.B. oxLDL) werden sie zu Schaumzellen, sezernieren inflammatorische Zytokine und befeuern somit die vaskuläre Entzündungsreaktion. Makrophagen können jedoch auch schützende Funktionen wahrnehmen, bspw. durch die antiinflammatorische Aufnahme apoptotischer Zellen, die Efferozytose. Um den Einfluss CD8+ T-Zellen auf Makrophagen zu bestimmen, wurde ein in vitro Model gewählt, in dem aktivierte CD8+ T-Zellen mit aus Knochenmark isolierten Makrophagen kokultiviert wurden. Zunächst konnte gezeigt werden, dass CD8+ T-Zellen die oxLDL Aufnahme und Schaumzellbildung der Makrophagen fördern, assoziiert mit der gesteigerten Expression des oxLDL Rezeptors CD36 und verminderten Expression des reversen Cholesterintransporters ABCA1. Zusätzlich reduzierten CD8+ T-Zellen die Phagozytose apoptotischer Zellen und die Sekretion des antiinflammatorischen Zytokins IL-10 als Antwort auf die Aufnahme apoptotischer Zellen, was auf eine verminderte Efferozytose hindeutet. Zudem förderten CD8+ T-Zellen die Expression des proinflammatorischen M1-Polarisationsmarker iNOS in Makrophagen und die Sekretion des proatherogenen Chemokins CCL2. Durch die Zugabe neutralisierender Antikörper in die in vitro Kultur konnte gezeigt werden, dass die aufgeführten Prozesse teilweise von den klassischen Effektorzytokinen der CD8+ T-Zellen, INFγ und TNFα, abhängen.
Zusammenfassend zeigen unsere Daten, dass CD8+ T-Zellen die Ausbildung eines proatherogenen Phänotyps der Makrophagen, durch die Steigerung der Schaumzellbildung und Förderung der proinflammatorischen Makrophagenpolarisation, sowie die Inhibierung der antiinflammatorischen Efferozytosefunktion, bewirken.
Sepsis ist eine dysregulierte Reaktion des Organismus auf eine Infektion. Bei Sepsis werden oft Blutungs- und Thromboseereignisse beobachtet, welche in einer Disseminierten Intravasalen Gerinnung (DIG) gipfeln können. Thrombozyten sind die Schlüsselzellen von Thrombose und Hämostase. Bei Sepsis und DIG kommt es häufig zu einem Abfall der Thrombozytenzahl, doch Blutungs- und Thromboseereignisse können unabhängig von der Thrombozytenzahl auftreten, was zusätzlich eine Veränderung der Thrombozytenfunktion nahelegt.
In dieser Arbeit wurde deshalb die Thrombozytenfunktion bei 15 Patienten mit Sepsis zu drei Zeitpunkten im Krankheitsverlauf untersucht. Es konnte bei unauffälliger Rezeptorexpression keine Voraktivierung der Thrombozyten mittels Durchflusszytometrie festgestellt werden. Jedoch war die Aktivierung nach Stimulation mit multiplen Agonisten signifikant reduziert. Besonders ausgeprägt war die Hyporeaktivität bei Stimulation des Kollagen-Rezeptors GPVI mit dem Agonisten CRP-XL. Es wurde gezeigt, dass nach GPVI-Stimulation eine reduzierte Phosphorylierung der nachgeschalteten Proteine Syk und LAT im Vergleich zum Gesundspender induziert wird. In Kreuzinkubationsexperimenten hatte die (Co )Inkubation von Thrombozyten in Plasma von Sepsispatienten oder mit Bakterienisolaten aus Sepsis-Blutkulturen keinen Effekt auf die Thrombozytenreaktivität. Allerdings konnte durch Sepsis-Vollblut eine signifikante GPVI-Hyporeaktivität in Thrombozyten von gesunden Probanden induziert werden, was einen zellulären Mediator als Ursache des Defekts nahelegt. In dieser Arbeit wurde gezeigt, dass insbesondere die GPVI-Signalkaskade bei Sepsis massiv beeinträchtigt ist. Der Immunorezeptor GPVI ist ein vielversprechendes Zielmolekül, um die Pathogenese der Sepsis, des Capillary Leak und die immunregulatorische Rolle von Thrombozyten besser zu verstehen. Die GPVI-Hyporeaktivität könnte als zukünftiger Biomarker für die Sepsis-Frühdiagnose genutzt werden.
Strumpellin is a member of the highly conserved pentameric WASH complex, which stimulates the Arp2/3 complex on endosomes and induces the formation of a branched actin network. The WASH complex is involved in the formation and stabilisation of endosomal retrieval subdomains and transport carriers, into which selected proteins are packaged and subsequently transported to their respective cellular destination, e.g. the plasma membrane. Up until now, the role of Strumpellin in platelet function and endosomal trafficking has not been researched. In order to examine its role, a conditional knockout mouse line was generated, which specifically lacked Strumpellin in megakaryocytes and platelets.
Conditional knockout of Strumpellin resulted in only a mild platelet phenotype. Loss of Strumpellin led to a decreased abundance of the αIIbβ3 integrin in platelets, including a reduced αIIbβ3 surface expression by approximately 20% and an impaired αIIbβ3 activation after platelet activation. The reduced surface expression of αIIbβ3 was also detected in megakaryocytes. The expression of other platelet surface glycoproteins was not affected. Platelet count, size and morphology remained unaltered. The reduction of αIIbβ3 expression in platelets resulted in a reduced fibrinogen binding capacity after platelet activation. However, fibrinogen uptake under resting conditions, although slightly delayed, as well as overall fibrinogen content in Strumpellin-deficient platelets were comparable to controls. Most notably, reduced αIIbβ3 expression did not lead to any platelet spreading and aggregation defects in vitro. Furthermore, reduced WASH1 protein levels were detected in the absence of Strumpellin.
In conclusion, loss of Strumpellin does not impair platelet function, at least not in vitro. However, the data demonstrates that Strumpellin plays a role in selectively regulating αIIbβ3 surface expression. As a member of the WASH complex, Strumpellin may regulate αIIbβ3 recycling back to the platelet surface. Furthermore, residual WASH complex subunits may still assemble and partially function in the absence of Strumpellin, which could explain the only 20% decrease in αIIbβ3 surface expression. Nonetheless, the exact mechanism still remains unclear.
Die Atherosklerose ist als Ursache kardiovaskulärer Erkrankungen, welche die häufigste Todesursache weltweit darstellen, von großer klinischer und wissenschaftlicher Relevanz. Atherosklerose ist charakterisiert durch Einlagerungen von Lipiden in die Gefäßwand, welche zur Ausbildung von Plaques führen. Als Folge wird eine chronische Entzündungsreaktion eingeleitet, die durch spezifische Immunzellen, unter anderem T-Lymphozyten, und komplexe molekulare Prozesse aufrechterhalten wird. Durch eine verminderte Sauerstoffdiffusionskapazität und eine hohe Zelldichte ist das Milieu in den Plaques hypoxisch. Zur zellulären Anpassung an ein solches hypoxisches Milieu werden Hypoxie-induzierbare Faktoren (HIF) in den Immunzellen stabilisiert. Der Transkriptionsfaktor HIF-1 ist ein heterodimeres Protein, welches die Transkription bestimmter Zielgene initiiert, die den Zellen notwendige Adaptationen des Zellstoffwechsels an ein vermindertes Sauerstoffangebot ermöglichen.
Das Ziel der vorliegenden Arbeit bestand darin zu untersuchen, inwiefern sich ein Ausschalten des Transkriptionsfaktor HIF-1α selektiv in T-Lymphozyten auf Atherosklerose und Myokardinfarkt auswirkt. Die funktionelle Bedeutung von HIF-1α in T-Zellen in der Pathogenese dieser Erkrankungen wurde an zwei Mausmodellen untersucht.
Im Atherosklerose Modell wurde Biomaterial von LDLR-/- Mäusen mit T-Zell spezifischem Knockout von HIF-1α nach achtwöchiger fettreicher Western-Typ Diät untersucht. Histologisch zeigte sich eine vermehrte Plaqueausprägung und ein verminderter Makrophagenanteil in den Plaques. Durchflusszytometrisch und mittels qPCR konnten keine Unterschiede in der Lymphozytendifferenzierung in Milz und Lymphknoten dieser Mäuse nachgewiesen werden.
Im Myokardinfarkt-Modell mit T-Zell spezifischem HIF-1α Knockout konnte in früheren Untersuchungen der Arbeitsgruppe eine vergrößerte Infarktzone mit eingeschränkter kardialer Funktion nachgewiesen werden. Histologisch konnte im Rahmen dieser Arbeit hierfür kein zellmorphologisches Korrelat in Kardiomyozytengröße oder der Vaskularisation des Myokards gefunden werden.
In Zukunft könnte HIF-1α in T-Lymphozyten ein möglicher Angriffspunkt zur medikamentösen Prävention oder Therapie kardiovaskulärer Erkrankungen sein.
In tumor therapy anti-angiogenic approaches have the potential to increase the efficacy of a wide variety of subsequently or co-administered agents, possibly by improving or normalizing the defective tumor vasculature. Successful implementation of the concept of vascular normalization under anti-angiogenic therapy, however, mandates a detailed understanding of key characteristics and a respective scoring metric that defines an improved vasculature and thus a successful attempt. Here, we show that beyond commonly used parameters such as vessel patency and maturation, anti-angiogenic approaches largely benefit if the complex vascular network with its vessel interconnections is both qualitatively and quantitatively assessed. To gain such deeper insight the organization of vascular networks, we introduce a multi-parametric evaluation of high-resolution angiographic images based on light-sheet fluorescence microscopy images of tumors. We first could pinpoint key correlations between vessel length, straightness and diameter to describe the regular, functional and organized structure observed under physiological conditions. We found that vascular networks from experimental tumors diverted from those in healthy organs, demonstrating the dysfunctionality of the tumor vasculature not only on the level of the individual vessel but also in terms of inadequate organization into larger structures. These parameters proofed effective in scoring the degree of disorganization in different tumor entities, and more importantly in grading a potential reversal under treatment with therapeutic agents. The presented vascular network analysis will support vascular normalization assessment and future optimization of anti-angiogenic therapy.
Introduction
Pro-thrombotic events are one of the prevalent causes of intensive care unit (ICU) admissions among COVID-19 patients, although the signaling events in the stimulated platelets are still unclear.
Methods
We conducted a comparative analysis of platelet transcriptome data from healthy donors, ICU, and non-ICU COVID-19 patients to elucidate these mechanisms. To surpass previous analyses, we constructed models of involved networks and control cascades by integrating a global human signaling network with transcriptome data. We investigated the control of platelet hyperactivation and the specific proteins involved.
Results
Our study revealed that control of the platelet network in ICU patients is significantly higher than in non-ICU patients. Non-ICU patients require control over fewer proteins for managing platelet hyperactivity compared to ICU patients. Identification of indispensable proteins highlighted key subnetworks, that are targetable for system control in COVID-19-related platelet hyperactivity. We scrutinized FDA-approved drugs targeting indispensable proteins and identified fostamatinib as a potent candidate for preventing thrombosis in COVID-19 patients.
Discussion
Our findings shed light on how SARS-CoV-2 efficiently affects host platelets by targeting indispensable and critical proteins involved in the control of platelet activity. We evaluated several drugs for specific control of platelet hyperactivity in ICU patients suffering from platelet hyperactivation. The focus of our approach is repurposing existing drugs for optimal control over the signaling network responsible for platelet hyperactivity in COVID-19 patients. Our study offers specific pharmacological recommendations, with drug prioritization tailored to the distinct network states observed in each patient condition. Interactive networks and detailed results can be accessed at https://fostamatinib.bioinfo-wuerz.eu/.
B cell maturation and immunoglobulin (Ig) repertoire selection are governed by expression of a functional B cell receptor (BCR). Naïve B cells co-express their BCR as IgM and IgD isotype. However, the role of the additionally expressed IgD on naïve B cells is not known. Here we assessed the impact of IgD on naïve B cell maturation and Ig repertoire selection in 8 individuals from 3 different families with heterozygous loss-of-function or loss-of expression mutations in IGHD. Although naïve B cells from these individuals expressed IgM on their surface, the IGHD variant in heterozygous state entailed a chimeric situation by allelic exclusion with almost half of the naïve B cell population lacking surface IgD expression. Flow cytometric analyses revealed a distinct phenotype of IgD-negative naïve B cells with decreased expression of CD19, CD20 and CD21 as well as lower BAFF-R and integrin-β7 expression. IgD-negative B cells were less responsive in vitro after engaging the IgM-BCR, TLR7/9 or CD40 pathway. Additionally, a selective disadvantage of IgD-negative B cells within the T2 transitional and mature naïve B cell compartment as well as reduced frequencies of IgMlo/- B cells within the mature naïve B cell compartment lacking IgD were evident. RNA-Ig-seq of bulk sorted B cell populations showed an altered selection of distinct VH segments in the IgD-negative mature naïve B cell population. We conclude that IgD expression on human naïve B cells is redundant for generation of naïve B cells in general, but further shapes the naive B cell compartment starting from T2 transitional B cells. Our observations suggest an unexpected role of IgD expression to be critical for selection of distinct Ig VH segments into the pre-immune Ig repertoire and for the survival of IgMlo/- naïve B cells known to be enriched in poly-/autoreactive B cell clones.
Zinc (Zn2+) is considered as important mediator of immune cell function, thrombosis and haemostasis. However, our understanding of the transport mechanisms that regulate Zn2+ homeostasis in platelets is limited. Zn2+ transporters, ZIPs and ZnTs, are widely expressed in eukaryotic cells. Using mice globally lacking ZIP1 and ZIP3 (ZIP1/3 DKO), our aim was to explore the potential role of these Zn2+ transporters in maintaining platelet Zn2+ homeostasis and in the regulation of platelet function. While ICP-MS measurements indicated unaltered overall Zn2+ concentrations in platelets of ZIP1/3 DKO mice, we observed a significantly increased content of FluoZin3-stainable free Zn2+, which, however, appears to be released less efficiently upon thrombin-stimulated platelet activation. On the functional level, ZIP1/3 DKO platelets exhibited a hyperactive response towards threshold concentrations of G protein-coupled receptor (GPCR) agonists, while immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptor agonist signalling was unaffected. This resulted in enhanced platelet aggregation towards thrombin, bigger thrombus volume under flow ex vivo and faster in vivo thrombus formation in ZIP1/3 DKO mice. Molecularly, augmented GPCR responses were accompanied by enhanced Ca2+ and PKC, CamKII and ERK1/2 signalling. The current study thereby identifies ZIP1 and ZIP3 as important regulators for the maintenance of platelet Zn2+ homeostasis and function.