Abstract
PL1 Coagulopathy and Anticoagulation in Covid-19: What can we learn for future challenges?
Daniel Duerschmied
Cardiology and Intensive Care Medicine, Heart Center, University of Freiburg, Germany
The Covid-19 pandemic has cost almost 4 million lives and taught us many lessons. A major challenge remains the coagulopathy, which comes with severe Covid-19. Traditional anticoagulation regimes are insufficient, because the rate of thrombotic complications ranges between 10% and 40%, despite state-of-the-art thrombo-prophylaxis with low-molecular weight heparins. Thrombotic complications are most frequent in severely sick patients with acute respiratory distress syndrome requiring intensive care. Bleeding complications on the other hand are also frequent in Covid-19 patients on intensive care units. It is therefore necessary to better understand the underlying mechanisms and find new therapeutic options.
SARS-CoV-2 enters the vasculature after leaving the alveolar space and causes endotheliitis (mostly) locally and a prothrombotic activation systematically. Endothelial cells, platelets, neutrophils and monocytes cooperate and use soluble factors such as tissue factor, neutrophil extracellular traps (NETs) and the complement system to induce thrombus formation in micro- and microvasculature. Among traditional anticoagulants, heparins are the generally recommended and most frequently used drugs. Considering newer insight into the pathomechanism of Covid-19 coagulopathy, targeting NETosis, heterocellular interactions, complement activation and endotheliitis appear however more promising strategies.
Vaccine-induced immune thrombotic thrombocytopenia (VITT) has been observed in approximately 15:100,000 cases of initial vaccination with ChAdOx1 nCoV-19 (AstraZeneca), a recombinant chimpanzee adenoviral vector encoding the spike protein of SARS-CoV-2. Thromboses occur often at unusual sites 5 to 24 days after vaccination, a typical time-course for immunogenic activation. Administration of a high dose of intravenous IgG is therefore a causal therapeutic option in confirmed cases.
PL2 Lessons learnt from comparative hemorheology
Ursula Windberger
Medizinische Universitaet Wien, Center for Biomedical Research
The animal kingdom offers numerous wildtype RBC phenotypes that enable us to study structurefunction relationships. But the species-specific hemorheological fingerprint forces us to carefully select the model for a particular research question. Although new data can be added to enhance knowledge on animal blood, some conclusions are not possible without the use of animal species. We show (1) that RBCs (like endothelial cells) possess a surface layer of species-specific thickness that protects the bilayer, attenuates the thermal input, and reduces the differences in RBC stiffness between species. This elastic halo not only increases the effective erythrocyte volume, but should also be considered for the coupling between RBCs and the flowing blood plasma. We further show that (2) the yielding of a blood suspension is not only a question of erythrocyte aggregation, (3) cytoplasmic elements (mitochondria, intermediate fibers, marginal bands) do not per se predict a high cell stiffness and flow resistance, and that (4) the bilayer area between the AE-1 anchor points to the cytoskeleton is important for cell membrane stabilisation.
PL3 On the path of cell biomechanics research
Masaaki Sato
Tohoku University
I started to go on the path to biomechanics research in my student days of Masters course in 1971. What is biomechanics? Fung defined “Biomechanics is mechanics applied to biology”. Surprisingly the term of “biomechanics” is not a new word, but had been used in Germany and Austria in 1800s. Typical examples of biomechanics I have been committed to are (1) mechanics of blood vessel wall, (2) blood flow dynamics in microvessels in the process of platelet thrombus formation, (3) mechanics of endothelial cells (EC) exposed to external forces. In my talk, I will mainly focus on the third topic, especially the effects of shear stress and hydrostatic pressure. My first path of cell biomechanics was measurement of mechanical properties of cultured EC exposed to fluid shear stress using micropipette technique. The mechanical stiffness significantly became greater than the control cell. The influence of shear stress on EC stiffness was related to changes in cytoskeletal structure. Since then, I have been interested in the mechanism of EC morphological change with cytoskeleton under mechanical forces. When external forces are loaded onto the cell during flow, force is considered to transmit via cytoskeletal elements to mechano-transduction sites such as receptors of cell membrane, junctions, focal adhesions and so on. The intracellular mechanical field was analyzed by observing deformation behaviour of living EC exposed to shear stress. Mechanical properties of stress fibers were also measured as a material for force transmission. Further, the effects of hydrostatic pressure on EC were investigated from a view point of mechanical adaptation and tissue activation.
PL4 Pathophysiology and treatment options for venous ulceration: Is there a role for exercise?
Markos Klonizakis
Lifestyle, Exercise and Nutrition Improvement (LENI) Research Group, Department of Nursing and Midwifery, Sheffield Hallam University, UK
Venous leg ulcers affect severely quality of life. They are associated with pain, restriction of work and leisure activities, impaired mobility and marked social isolation. Although most of them heal within 3 months, it can take up to 24 weeks or 51 visits for leg ulcers to heal. The occurrence of venous leg ulcers is strongly linked to advanced age: it is estimated that the prevalence in the over-65-year age group is about 3%. Considering also that they can cost up to €2.4 k/year each, it can be agreed that they pose a costly and relatively common health condition with serious societal effects. This plenary lecture will discuss the different leading theories for the pathophysiology of venous leg ulceration, present the main current therapeutic pathways and finally discuss the most recent findings on the use of exercise as an adjunctive therapy for this condition, exploring the role that physical activity and exercise may have as treatment options.
PL5 Hemodynamic functionality of transfused red blood cells: A potent effector of transfusion outcome
Saul Yedgar1, Neta Goldschmjdt2, Gregory Barshtein1
1 The Hebrew University Medical School, Jerusalem, Israel, 2 Hadasah Hospital, Jerusalem, Israel
Red blood cell (RBC) transfusion is aimed at supplying oxygen to tissues. However, RBCs have unique flow affecting properties, specifically deformability, adherence to vascular endothelial cells and aggregability, which define their hemodynamic functionality (HF), namely their potential to affect blood circulation. Numerous studies, reporting adverse transfusion outcomes, including impaired blood perfusion, have raised a growing concern about the risks in blood transfusion. To test the role of the HF of transfused RBC (T-RBC) in transfusion outcome, we examined its effect on the transfusion-induced change in the skin blood flow and hemoglobin increment, in β-thalassemia major patients, who are treated with life-long frequent blood transfusion (every 2–4 weeks). The results showed, for the first time in humans, that the HF of T-RBC is a potent effector of transfusion outcome: The transfusion-induced change in the recipients’ blood circulation increased when the HF of the T-RBC was better than that of the recipients, but decreased when the HF of the recipients’ RBC was better than that of the TRBC. The transfusion-induced Hb increment increased as function of the T-RBC HF. Of special interest the time interval between consecutive transfusion increased with increasing HF of the T-RBC (less transfusions required), suggesting a link between the HF of the transfused RBC and their survival in the recipients’ vascular system. The current tests of blood units focus on immunological compatibility and pathogenic agents, while the functionality of the T-RBC is ignored. Monitoring the HF of T-RBC would make paradigm shift in blood banking, which would reduce the risk in blood transfusion and improve its outcome.
PL6 Lessons from red blood cell mechanics to endothelial cell mechanobiology
Kris Dahl
Carnegie Mellon University
Decades of study of the erythrocyte membrane mechanics, structure of the actin-spectrin skeleton and internal rheology have provided a wealth of information performed on a simple cellular system. Over the last twenty years the mechanical and rheological properties of nucleated cells has been shown to be important in mechano-transmission (the movement of mechanical force) as well as mechano-biology (the processing of mechanical force to biological properties). Many of the same fundamental aspects learned from red blood cells have been applied to endothelial cells. For example, we have found spectrins at the plasma membrane and in the nucleus. These spectrins aid in mechano-transmission as tension regulators and springs within the cell. We also measure rheological changes inside endothelial cell nuclei associated with shear stress, aging and growth factors to consider mechano-biology. Unlike red cells, endothelial cells have changes in DNA compaction, gene expression as well as molecular motor forces. We are able to parse out contributions of these different rheological contributions to nuclear rheology to better quantify mechano-biology within the endothelial cell.
PL7 Emerging roles of membrane lipids and mitochondria in endothelial cell mechano-sensing
Kimiko Yamamoto
The University of Tokyo
Vascular endothelial cells (ECs) sense hemodynamic shear stress and transduce this information into functional responses, which are critical for circulatory homeostasis and the development of various vascular diseases, including hypertension, thrombosis, and atherosclerosis. A number of studies have shown that when shear stress acts on ECs, a wide variety of signalling pathways are activated almost simultaneously through various membrane molecules and micro-domains such as ion channels, receptors, adhesion molecules, caveolae, and primary cilia. However, the specific underlying mechanisms responsible for such mechano-sensing remain elusive. Recently, plasma membranes and mitochondria have emerged as new players in EC mechano-sensing. Application of shear stress to ECs or artificial lipid bilayers immediately decreased the lipid order and increased the fluidity of the membrane, while another hemodynamic force, stretching tension, induced the opposite changes. These changes in membrane physical properties were linked downstream to membrane receptor activation specific to shear stress and stretch, i.e. phosphorylation of PDGF receptors and phosphorylation of VEGF receptors, respectively. Thus, plasma membranes appear to act as a mechano-sensor that is capable of differentiating between shear stress and stretch, thereby leading to mechano-transduction specific to each force. In addition, imaging with a newly developed cholesterol biosensor showed that shear stress rapidly reduced cholesterol in both the outer and inner leaflets of the plasma membrane. Furthermore, real-time imaging of ATP inside mitochondria revealed that exposure of ECs to shear stress augmented mitochondrial ATP generation, triggering ATP release to the extracellular space and subsequent activation of EC purinoceptors leading to Ca2+ signalling. Similarly, the treatment of ECs with methyl-β-cyclodextrin, a membrane cholesterol-depleting agent, augmented mitochondrial ATP production, suggesting that plasma membrane cholesterol dynamics are closely coupled to mitochondrial oxidative phosphorylation in ECs. Elucidation of this linkage between membrane lipids and mitochondria would lead to a better understanding of EC mechano-sensing.
PL8 The mechano-transduction of cancer and blood cells exposed to circulatory levels of fluid shear stress
Michael R. King
Vanderbilt University
TRAIL specifically induces apoptosis in cancer cells without affecting healthy cells. However, TRAIL’s cancer cytotoxicity was insufficient in clinical trials. Circulatory-shear stress is known to sensitize cancer cells to TRAIL. In our laboratory, we have studied the mechanism of this TRAIL sensitization with the goal of translating it to static conditions. GsMTx-4, a Piezo1 inhibitor, was found to reduce shear stress-related TRAIL sensitization, implicating Piezo1 activation as a potential TRAIL-sensitizer. The Piezo1 agonist Yoda1 recreated shear stress-induced TRAIL sensitization under static conditions. A significant increase in apoptosis occurred when prostate, colon, or breast cancer cells were treated with Yoda1 and TRAIL in combination, but not in Bax-deficient cells. Cells exposed to fluid shear and TRAIL showed increased mitochondrial outer membrane permeability (MOMP), mitochondrial depolarization, and activated Bax. This implies that Piezo1 activation sensitizes cancer cells to TRAIL through a calcium influx that activates calpains. A computational model has been used to elucidate the proapoptotic or antiapoptotic roles of Bax, Bcl-2, XIAP, and other proteins important in the mitochondrial-apoptotic signalling pathway. In separate studies, we have found that the activation of T cells via CD3 and CD28 antibodies can be greatly enhanced with fluid shear stress, which may have important implications for future immunotherapy applications.