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The endothelial glycocalyx plays an important role as part of the permeability barrier between the blood and the interstitium. In this study, we used different sized fluorescently labeled dextran molecules to determine the size of the macromolecular exclusion zone in capillaries. The width of the exclusion zone was calculated as one half the difference between the anatomic luminal diameter, as determined by transillumination, and the width of a fluorescent dextran column. During the first hour after systemic injection of labeled dextrans, neither 70 kDa dextran (Dextran 70) nor 500 kDa dextran (Dextran 500) labeled with the anionic fluorescein isothiocyanate (FITC) penetrated the endothelial glycocalyx to the endothelial cell surface. However, the 40 kDa dextran (Dextran 40) labeled with the neutral fluorophore Texas Red was able to penetrate to the endothelial cell surface. Under these control conditions, the width of the exclusion zone for Dextran 500 was 0.55 ± 0.02 mm (n=46); for Dextran 70 it was 0.50 ± 0.01 mm (n=111); and for Dextran 40 it was 0.08 ± 0.01 mm (n=53). One hour after systemically injecting the enzyme hyaluronidase, measurements of the exclusion zone were made using Dextrans 40, 70 and 500. After the enzyme treatment, Dextran 70 appeared to penetrate the glycocalyx layer, whereas Dextran 500 did not. Following hyaluronidase treatment, the width of the exclusion zone for Dextran 500 was 0.56 ± 0.02 mm (n=71); for Dextran 70 it was 0.05 ± 0.01 mm (n=103); and for Dextran 40 it was 0.03 ± 0.01 mm (n=33). These results indicate that the enzyme hyaluronidase was able to degrade the structural integrity of the glycocalyx, since after enzymatic treatment Dextran 70 was able to permeate the glycocalyx layer, while it was unable to prior to this treatment. However, the glycocalyx barrier was not completely compromised following hyaluronidase treatment since Dextran 500 still was not able to permeate the exclusion zone. In conclusion, macromolecules with 5.3 nm or larger radii will more than likely not be able to permeate an intact glycocalyx; in addition, degradation of hyaluronan will increase the permeability of the glycocalyx so that macromolecules with 5.3 nm radii will permeate.

Proefschrift Universiteit van Amsterdam. / Met lit. opg. - Met samenvatting in het Nederlands.

Proefschrift Universiteit van Amsterdam. / Met een samenvatting in het Nederlands.

Trotz enormer Fortschritte in der Therapie, bleibt der kardiogene Schock die führende Todesursache im akuten Myokardinfarkt. Die pathophysiologischen Veränderungen umfassen dabei unter anderem Störungen der Mikrozirkulation, endotheliale Dysfunktion mit vaskulärer Leckage, sowie vermehrte Thrombozyten- und Leukozytenadhäsion an die Gefäßwand. Die endotheliale Glykokalyx wurde als zentraler Regulator dieser Prozesse identifiziert. Das Glykosaminoglykan Heparansulfat repräsentiert dabei den Hauptbestandteil der Endothelzelloberfläche und Syndecan-1 das am weitesten verbreitete Proteoglykan. Diesbezüglich konnte in Studien eine Assoziation zwischen Schädigung der endothelialen Glykokalyx und den zirkulierenden Membranbestandteilen im Patientenblut beobachtet werden. Ziel der Arbeit war die Analyse der Glykokalyxmarker bei 184 Patienten mit Infarkt-assoziiertem kardiogenen Schock. In den Serumproben zum Zeitpunkt der Aufnahme und nach einem Tag wurde mittels ELISA die Konzentration von Heparansulfat und Syndecan-1 bestimmt. Dabei zeigte sich ein signifikanter Konzentrationsabfall von Syndecan-1 innerhalb des Analysezeitraums. Des Weiteren hatten die Überlebenden an beiden Tagen signifikant niedrigere Syndecan-1-Serumwerte. Durch eine schrittweise Multiregressionsanalyse wurde Syndecan-1 bei Patienten mit akutem Myokardinfarkt und assoziiertem kardiogenen Schock als unabhängiger Prädiktor der 30-Tage- Mortalität identifiziert.

Cardiovascular disease is the leading cause of unnatural death worldwide. Damage to the endothelial glycocalyx impairs endothelial functions and thereafter leads to the development of cardiovascular diseases. Despite this, many issues remain to be explored in our understanding of the metabolism and vasculoprotective potential of the glycocalyx. This study focuses on the recovery and structural stability of the glycocalyx, and its role in electric field-directed cell migration in vitro. The integrity of the glycocalyx is compromised following trypsin treatment during cell passages. Results from our study show that cell seeding density affects the recovery speed of the glycocalyx in the first 48h. Higher cell density results in more rapid recovery of the glycocalyx. Regardless of the initial cell seeding density, a well-developed glycocalyx layer is observed when cell confluence is reached. Micropatterning is used to study effects of the cell shape on the recovery of the glycocalyx. Elliptical patterns have been used to conform endothelial cells to torpedo shapes, mimicking their morphology under a shear flow. More rapid development of the glycocalyx on elliptical cells is observed than that on circular shaped cells during the early stage of recovery. Effects of the actin cytoskeleton on the stability of the glycocalyx are investigated, following our interest in shedding of the glycocalyx in abnormal vascular microenvironment. Rapid depolymerisation of the actin cytoskeleton leads to cell retraction within 10mins, with the glycocalyx preserved on the cell surface. This is also seen during 24h persistent actin disruption under static conditions. However, when endothelial cells are subjected to 24h steady laminar shear stress, the glycocalyx is seen to shift to the downstream of the cell surface in the control group, and with actin depolymerisation, significant shedding of the glycocalyx from the luminal surface of the cell is observed. This happens together with the loss of focal adhesions on the basal membrane. Using a custom designed electric field (EF) chamber, I demonstrate that the cell migration speed increases by 30~40% following 5h of EF exposure. Cells also show preferred movement towards the anode. However, both are abolished after the enzymatic removal of the glycocalyx, indicating that the speedup and the directional cell migration in applied EF require the presence of the glycocalyx. Even distribution of the glycocalyx on the cell surface at the end of the EF stimulation suggests that EF-directed cell migration is not related to the polarization of the glycocalyx on the cell membrane. All these findings provide a better understanding of the glycocalyx, which will help to develop new strategies for protection of the glycocalyx, restoration of endothelial functions and finally prevention of cardiovascular diseases.

Excessive microvascular permeability is a serious complication involved in traumatic injury and inflammatory diseases. Alcohol intoxication can exacerbate the physiological derangements produced by microvascular endothelial barrier dysfunction in such disease conditions. Sphingosine-1-phosphate (S1P) has known endothelial barrier-protective properties, and has been shown to ameliorate microvascular leakage in a model of combined alcohol intoxication and hemorrhagic shock and resuscitation (HSR). However, whether the barrier-protective properties of S1P extend to endothelial cells of the blood-brain barrier (BBB) is unclear. The mechanisms of S1P-induced barrier protection during alcohol intoxication or HSR are also unknown. In the current study, we tested the hypothesis that S1P could enhance endothelial barrier during alcohol intoxication or hemorrhagic shock by preserving the integrity of junction proteins and the endothelial glycocalyx, and protecting mitochondrial function. Cultured primary human brain microvascular endothelial cell (HBMEC) monolayers were used to characterize endothelial-specific mechanisms of S1P protection of the BBB during alcohol treatment. Transendothelial electrical resistance (TER) and apparent permeability coefficients for albumin, dextran-4 kDa, and sodium fluorescein were used as indices of barrier function. Junctional localization was determined by immunofluorescence confocal microscopy. We also used an established in vivo rat model of conscious HSR and assessed microvascular leakage, endothelial glycocalyx integrity, and mitochondrial function by intravital microscopy. Cultured rat intestinal microvascular endothelial cell (RIMEC) monolayers were used to test the ability of S1P to protect against glycocalyx shedding and endothelial barrier dysfunction caused by direct disruption of mitochondrial integrity due to inhibition of mitochondrial complex III. The results show that alcohol significantly impaired HBMEC TER and increased solute permeability, which was reversed with application of S1P after alcohol treatment. Alcohol caused the formation of gaps between cells. Treatment with S1P (after alcohol) increased junctional localization. Our in vivo results show that S1P protects against HSR-induced hyperpermeability, preserves the expression of adherens junctional proteins, and protects against glycocalyx degradation. S1P treatment during HSR also protects against mitochondrial membrane depolarization. Besides that, S1P protects RIMECs against mitochondrial dysfunction-induced endothelial barrier dysfunction and glycocalyx degradation by acting through mitochondrial complex III. Our results indicate that S1P may be useful for restoring BBB function during alcohol intoxication. Moreover, S1P protects against HSR-induced mitochondrial dysfunction in endothelial cells, which in turn improves the structure of the endothelial glycocalyx after HSR and allows for better junctional integrity to prevention of excess microvascular permeability.

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.

<p>Complications from cardiovascular disease, such as a heart attack or a stroke, represent the leading cause of death in the United States and many developed and developing countries. Atherosclerosis is the primary pathology underlying cardiovascular disease. It is caused by an increase in endothelial cell (EC) permeability, leading to the buildup of cholesterol and lipids which form the atherosclerotic plaque. Accelerated aging in regions of atherosclerosis contribute to the development and progression of the disease. The global hypothesis that motivated this research was that activation of deacetylase antioxidant regulator, Sirtuin1 (SIRT1), improved function in senescent endothelial cells ECs by increasing the integrity of cell-cell junctions. In turn, this led to elevated EC permeability, a decline in the response to shear stress, and elevated traction forces.</p><p>Aging of hCB-ECs significantly increased permeability due to changes in tight junction protein localization and phosphorylation. Activation of SIRT1 significantly reduced permeability in aged hCB-ECs and restored junction integrity. ECs under flow also exhibited changes in cell junctions with increasing age. Aged hCB-ECs were less responsive to shear stress, exhibiting lower levels of atheroprotective genes, KLF2 and eNOS. Activation of SIRT1 in aged hCB-ECs restored the response to shear stress by altering localization and phosphorylation of adherens junction protein, VE-cadherin. The endothelial glycocalyx is a layer of proteoglycans and glycoproteins on the surface of ECs that is important in maintaining EC barrier function. In aging ECs, the glycocalyx was thinner and less dense. However, activation of SIRT1 restored the structure of the glycocalyx, suggesting that the positive effect of SIRT1 on elevated permeability in aged hCB-ECs may also be due to restoration of the glycocalyx. Aged hCB-ECs also exhibited elevated traction forces for measurements done with single cells, cell clusters (2 to 3 cells), and cell monolayers (20 to 30 cells). The elevated traction forces correlated with altered actin localization and increased actin filament thickness. Activation of SIRT1 reduced traction forces and decreased actin stress fiber thickness in aged hCB-ECs, suggesting that the effects of SIRT1 on cell-cell junctions alters binding of junction proteins to the actin cytoskeleton and generation of cell traction forces. Together, these results implicate an important role for SIRT1 in regulating permeability and mechanotransduction in aging endothelium.</p> / Dissertation

Myelin ist eine mehrschichtige Membran, die die Axone in peripheren (PNS) und Zentrale Nervensystem (ZNS) umhüllt. Die Bildung und Anordnung dieser Struktur ist ein mehrstufiger Prozess, der durch eine Vielzahl extrazellulärer Faktoren reguliert wird. Im ZNS wird Myelin von Oligodendrozyten gebildet. Während der Entwicklung differenzieren die Vorläufer dieser Zellen zu reifen Oligodendrozyten aus. Nachdem sie das geeignete Signal aus ihrer Umgebung erhalten haben, beginnen die Oligodendrozyten die Axone mit Myelinmembranen einzuhüllen.  Allerdings sind die Signale, die diesen Prozess initiieren unbekannt. Mit dieser Arbeit zeigen wir, dass Oligodendrozyten kleine Mikrovesikel - so genannte Exosomen - in den extrazellulären Raum freisetzen, welche die terminale Differenzierung von Oligodendrozyten und die anschließende Myelinbildung verhindern. Es konnte gezeigt werden, dass diese inhibitorische Wirkung durch die Aktivität der RhoA-ROCK-Signalkaskade vermittelt wird. Bemerkenswerterweise war die Exosomenfreisetzumg durch Oligodendrozyten signifikant reduziert, wenn die Zellen mit konditioniertem Medium von Neuronen inkubiert wurden. Unsere Ergebnisse legen nahe, dass Exosomen, die von Oligodendrozyten produziert werden,  Zellen in einem pre-myelinisierten Stadium halten, während die Sekretion von Exosomen in Gegenwart neuronaler Signale reduziert wird und autoinhibitorische Signale aufgehoben werden. Somit können Neuronen die Bildung und Freisetzung von Exosomen regulieren, welche von Oligodendrozyten freigesetzt werden, um die Biogenese und Assemblierung der Myelinmembran zu koordinieren.  Im zweiten Teil der Arbeit wurde die Frage, wie die Kompaktierung des Myelins vermittelt wird, erörtert. Während bekannt ist, dass MBP die Interaktion zwischen Myelinmembranen von cytoplasmatischer Seite aus organisiert, ist der zugrundeliegende molekulare Mechanismus der Interaktion zwischen den äußeren Membranen nach wie vor unklar. Im Allgemeinen erfordert die Interaktion zwischen zwei gegenüberliegenden Membranen die Expression von Adhäsionsmolekülen und die Entfernung von repulsiven Komponenten. Daher untersuchten wir die Rolle des Proteolipid-Proteins (PLP), als mutmaßliches Adhäsionsmolekül, und die Glykocalix, als repulsive Struktur während der Myelinkompaktierung im ZNS. Wir analysierten die Adhäsion von aufgereinigten Myelinpartikeln mit den primären Oligodendrozyten, um die Wechselwirkung zwischen den Myelinschichten zu imitieren. Mit diesem System haben wir gezeigt, dass PLP die Adhäsionsfähigkeit der Myelinmembran erhöht. Mittels Single Particle Force-Spektroskopie fanden wir außerdem heraus, dass PLP die physikalische Stabilität von Myelin verbessert. Zusätzlich beobachteten wir eine signifikante Reduzierung in der Glykokalix während der Oligodendrozytenreifung, die mit einer Zunahme in ihrer Oberflächenaffinität gegenüber den Myelinpartikeln korreliert. Weitere Analysen zeigten, dass die negative Ladung der Zuckeranteile, hauptsächlich der Sialinsäure, für die Verringerung der Myelinadhäsion verantwortlich ist. Daher schlagen wir vor, dass die Adhäsionseigenschaften von PLP zusammen mit der Reduzierung der Glykokalyx, die Adhäsion der Myelinmembran und die  Kompaktierung im ZNS organisieren.