Transcapillary exchange is the movement of fluid and molecules through the porous capillary wall, and is important in maintaining homeostasis of bodily tissues. The classical view of this process is that of Starling's hypothesis, in which the forces driving filtration or absorption are the hydrostatic and osmotic pressure differences across the capillary wall. However, experimental evidence has emerged suggesting the importance of the capillary wall ultrastructure, and thus rather than the global differences between capillary and tissue, it is the local difference across a structure lining the capillary wall known as the endothelial glycocalyx that determines filtration. Hu and Weinbaum presented a detailed cellular level microstructural model of this phenomenon which was able to explain some experimental discrepancies. In this Thesis, rather than describing the microstructural details, the capillary wall is treated as a poroelastic material. The assumptions of poroelasticity theory are such that the detailed pore structure is smeared out and replaced by an idealized homogeneous system in which the fluid and solid phases coexist at each point. The advantage of this approach is that the mathematical problem is greatly simplified such that analytical solutions of the governing equations may be obtained. This approach also allows calculation of the stress and strain distribution in the tissue. We depart from classical poroelasticity, however, due to the fact that since there are concentration gradients within the capillary wall, the filtration is driven by both hydrostatic and osmotic pressure gradients. The model predictions for the filtration flux as a function of capillary pressure compares favourably with both experimental observations and the predictions of the microstructural models. An important factor implicated in transcapillary exchange is the endothelial glycocalyx, which was shown experimentally to protect against edema formation. Using our theory in combination with the experimental measurements of glycocalyx thickness and pericapillary space dimension (PSD), we make a quantitative comparison for the excess flow as a result of a deteriorated glycocalyx, which shows reasonably good agreement with the data. Since many of the parameters in the model are difficult to measure, a sensitivity analysis was performed on the most important of these. Finally, since there was variation in the measurements of glycocalyx thickness and PSD, we used probability distributions to represent the data, and performed further calculations to obtain ranges of likely values for the various parameters. This work could find applications in cardiovascular disease, where the glycocalyx is degraded or absent, and in cancer research, where the abnormal vasculature is an impediment to the efficient delivery of anti-cancer drugs.

Transcapillary exchange is the movement of fluid and molecules through the porous capillary wall, and is important in maintaining homeostasis of bodily tissues. The classical view of this process is that of Starling's hypothesis, in which the forces driving filtration or absorption are the hydrostatic and osmotic pressure differences across the capillary wall. However, experimental evidence has emerged suggesting the importance of the capillary wall ultrastructure, and thus rather than the global differences between capillary and tissue, it is the local difference across a structure lining the capillary wall known as the endothelial glycocalyx that determines filtration. Hu and Weinbaum presented a detailed cellular level microstructural model of this phenomenon which was able to explain some experimental discrepancies. In this Thesis, rather than describing the microstructural details, the capillary wall is treated as a poroelastic material. The assumptions of poroelasticity theory are such that the detailed pore structure is smeared out and replaced by an idealized homogeneous system in which the fluid and solid phases coexist at each point. The advantage of this approach is that the mathematical problem is greatly simplified such that analytical solutions of the governing equations may be obtained. This approach also allows calculation of the stress and strain distribution in the tissue. We depart from classical poroelasticity, however, due to the fact that since there are concentration gradients within the capillary wall, the filtration is driven by both hydrostatic and osmotic pressure gradients. The model predictions for the filtration flux as a function of capillary pressure compares favourably with both experimental observations and the predictions of the microstructural models. An important factor implicated in transcapillary exchange is the endothelial glycocalyx, which was shown experimentally to protect against edema formation. Using our theory in combination with the experimental measurements of glycocalyx thickness and pericapillary space dimension (PSD), we make a quantitative comparison for the excess flow as a result of a deteriorated glycocalyx, which shows reasonably good agreement with the data. Since many of the parameters in the model are difficult to measure, a sensitivity analysis was performed on the most important of these. Finally, since there was variation in the measurements of glycocalyx thickness and PSD, we used probability distributions to represent the data, and performed further calculations to obtain ranges of likely values for the various parameters. This work could find applications in cardiovascular disease, where the glycocalyx is degraded or absent, and in cancer research, where the abnormal vasculature is an impediment to the efficient delivery of anti-cancer drugs.

Vascular diseases, such as stroke and heart attacks, account for more than 50% of abnormal death worldwide. The cause of these diseases is linked to malfunctions of vascular endothelial cells, in particular the endothelial glycocalyx. This study investigates the location and stability of the endothelial glycocalyx under different flow conditions in vitro. AFM (Atomic Force Microscopy) micro indentation is carried out on endothelial cell membrane to determine its Young's modulus. The Young's modulus of the glycocalyx layer is then deduced from measurements on cell membranes with, and those without, the glycocalyx layer. Heparan sulphate (HS) is an important component of the glycocalyx and can be removed by the enzyme heparinase-III (Hep-III). Our results show the glycocalyx on cultured Human Umbilical Vein Endothelial Cells (HUVECs) has a Young's modulus of ~0.64Kpa. We further observe how the Young's modulus of the endothelial cell membrane decreases with time, as the glycocalyx layer redevelops, following its removal by Hep-III. Steady and oscillatory shear stimulations are used in flow chamber experiments. Under 24 hours' steady shear stimulation (12.6 dyn/cm2), cells are seen to elongate and reorient parallel to the flow direction. The glycocalyx is seen to shift to the peripheral region of the cell surface. With actin depolymerisation treatment, significant shedding of the glycocalyx from the luminal surface of the cell is observed. This occurs together with the loss of focal adhesions on the basal membrane. When endothelial cells are subjected to 24 hours' oscillating shear stress, the size of the cell increases as the oscillatory reversal time (time between changes in oscillatory flow direction) increases. Measurements are taken with oscillatory flow reversal programmed at 5s, 10s and 15s. The angle (between the long axis of the cell and the flow direction) and the aspect ratio (long axis vs short axis) change from 41.57° and 1.72 : 1 (static) to 40.18° and 3.26 : 1 (5s), 36.71° and 4.17 : 1 (10s), 26.5° and 4.39 : 1 (15s). Both the height and the area of the cell increase. The Young's modulus of the endothelial cell membrane is measured under oscillatory flows with different reversal time and compared to that under static flow conditions. An increase in the Young's modulus is observable under oscillatory flows, with the most significant change occurring at the edge (i.e. periphery) of the cell membrane area. As the oscillatory reversal time increases from 5s to 15s, the Young's modulus of the cell membrane increases. In the apical areas of the cell membrane, the increase is less significant. These results indicate that the thickness of the glycocalyx decreases as cells are exposed to oscillatory flows, and the loss is most significant in the peripheral region of the cell membrane. As the oscillatory reversal time increases from 5s to 15s, so the loss in the glycocalyx increases.

UNIVERZITA KARLOVA Lékařská fakulta v Plzni Dizertační práce Endothelial glycocalyx - diagnostic approach and intervention assessment MUDr.Jiří Pouska ABSTRACT Endothelial glycocalyx (EG) is fine structure on the surface of endothelium. After extensive research in past years, revisited Starling principle was finally formulated. It describes fluid physiology in capillaries precisely. EG has pivotal role in keeping endothelium semipermeable and thus avoiding extensive filtration of fluids to interstitium. Assessment of EG is clinically difficult. Many pathological conditions lead to damage of EG (sepsis etc.). Intravenous fluid therapy is mainstay of treatment of such conditions. Our aim was to determine the changes of EG integrity depending on the choice of intravenous fluid and its infusion time in physiological and pathological conditions. Key words: Endothelial glycocalyx, infusion therapy, anaesthesia, sepsis, microcirculation.

Une brosse de polymères est une matrice dense de macromolécules greffées à une surface donnée. Au-delà des brosses synthétiques réalisées en laboratoire, on trouve des exemples très variés dans la nature: un exemple emblématique est le glycocalyx endothélial, décorant la surface interne des vaisseaux sanguins des mammifères. L'interaction de cette structure avec le plasma et les cellules sous écoulement n'est encore que très partiellement explorée. La présente thèse propose, grâce à des simulations de "Dissipative Particle Dynamics", un modèle coarse-grained pour une analyse auto-cohérente d'une brosse polymérique dense sous écoulement parabolique. Cette étude mésoscopique met en évidence l'importance des effets collectifs entre molécules, entraînée par l'hydrodynamique, et propose des nouvelles interprétations à la phénoménologie du système brosse-écoulement. Des résultats préliminaires sont également produits pour l'interaction sous écoulement entre un objet mésoscopique déformable (prototype d'un globule rouge) et les polymères greffés. / Polymer brushes are dense matrices of grafted macromolecules. In addition to brushes finely designed in laboratory, various examples are offered by Nature, as the endothelial glycocalyx, decorating the lumen of mammalian blood vessels. The interaction of such network with the flowing plasma and cells is still partially unknown.The present thesis, by mean of Dissipative Particle Dynamics simulations, proposes a coarse-grained model for the self-consistent analysis of a dense polymer brush under parabolic flow. Our mesoscale investigation highlights the relevance of collective effects, driven by hydrodynamics, and proposes novel interpretations regarding the rich phenomenology of the brush-flow system.Preliminary results are also provided for the interplay between a mesoscopic deformable flowing object (prototype of a red blood cell) and the grafted polymers.