In den letzten Jahren gab es Fortschritte im Verständnis der Gefäßbildung bei Ereignissen wie Keimen, Lumenbildung und Gefäßstabilisierung. Nach der Bildung eines primitiven Plexus ist die Gefäßoptimierung und hierarchische Umwandlung der morphologischen Gefäße in einen reifen Plexus durch vaskuläres "Pruning" wenig verstanden. Unterschiedliche Blutflussprofile in nebeneinander angeordneten Gefäßen können Asymmetrien in der Scherspannung verursachen, was die Zellmigration in Bereichen mit höherem Fluss fördert und die Destabilisierung von Segmenten mit geringem Fluss induziert. Diese Studie basiert auf der Hypothese, dass funktionelle Gefäßnetzwerke und Umbildung durch flussgesteuerte Endothelzellmigration ausgelöst werden. Wie die zelluläre Erfassung physikalischer Kräfte integriert ist, um Informationen zu übertragen und das Verhalten von Zellen zu modifizieren, ist noch unbekannt. Die Studie untersucht die Regulation und Koordination durch RhoGTPase-Signale während der kollektiven endothelialen Migration aufgrund von Flüssigkeitskräften. RhoGTPasen ermöglichen die räumlich-zeitliche Koordination, langfristige Anpassung an den Fluss und morphologische Umgestaltung. Beeinträchtigungen von RhoGTPasen zeigen Defekte bei Zellmigration und kollektiver Koordination in Umgebungen mit freiem Rand und strömungsgetriebener Migration. Die Studie erläutert den Einfluss der RhoGTPase-Regulation der Verbindungsdynamik in Verbindung mit der Aktinorganisation, die für die mechanische Kopplung und endotheliale Reaktionsfähigkeit erforderlich ist. Insgesamt betont die Studie die Relevanz der räumlich-zeitlichen RhoGTPase-Kontrolle und der Aufrechterhaltung der mechanischen Kopplung zwischen Strömung und Migration für die kollektive Koordination als Reaktion auf hämodynamische Kräfte. / In recent years, there have been significant advances in understanding how new vessels form during events like sprouting, lumen formation, and vessel stabilization. Yet, after the formation of a basic network, the crucial step of rearranging vessels into a mature structure, known as vascular pruning, needs further investigation. It's suggested that different blood flow profiles in nearby vessels create imbalances in shear stress, leading to cell migration toward higher flow regions, destabilizing low-flow segments, and causing the collapse of redundant segments. This study, in line with existing literature, proposes that functional vascular networks and remodeling result from the flow-driven migration of endothelial cells. However, how cells precisely sense physical forces to regulate their behavior and coordinate migration in response to flow remains unknown. I explore the regulation and coordination of Rho GTPases during collective endothelial migration under fluid forces. Rho GTPases' coordination allows long-term adaptation to flow and morphological remodeling. Impairments in Rho GTPases reveal defects in cell migration and collective coordination during free-edge and flow-driven migration. Finally, I explain how Rho GTPases' regulation influences junctional dynamics and actin organization, crucial for mechanical coupling and endothelial responsiveness to flow. Overall, this study emphasizes the importance of controlling Rho GTPases over time and maintaining mechanical coupling between flow and migration for collective coordination in response to fluid forces.

Einleitung: Die Wundheilung kornealer Endothelzellen erfolgt hauptsächlich durch Migration benachbarter Zellen. Die Endothelzellen sind in vivo ständig in Kontakt mit Kammerwasser (KW). TGF-ß2 und Ascorbin- säure (AS) sind in hoher Konzentration im KW enthalten. Der Einfluß von humanem KW, TGF-ß2 und AS auf Proliferation und Migration boviner kornealer Endothelzellen (BCEC) in Kultur wurde untersucht. Methoden: Proliferation: BCEC der 1.Passage wurden zu 1,5x104 Zellen/Well ausgesät. Mit frischem Medium und Zusatz von humanem KW (10% und 100%), TGF-ß2 (0,1; 1 und 10 ng/ml) und AS (50, 100 und 200 µg/ml) wurden die Kulturen für 72 oder 96 h inkubiert und anschließend gezählt. Migration: Konfluente Zellkulturen der 1.Passage wurden verwendet. Mit einem modifizierten Trepan (Durchmesser 5,5 mm) wurde eine zentrale, zirkuläre "Wunde" gesetzt. Die Kulturen wurden mit frischem Medium, das humanes KW, TGF-ß2 und AS in den o. g. Konzentrationen enthielt, für 72 oder 96 h inkubiert. Zur Auswertung wurden die in den Wundbereich migrierten Zellen an 5 verschiedenen Stellen, ausgehend vom Wundrand, gezählt. Ergebnisse: Die Proliferation der BCEC wurde durch humanes KW (unverdünntes KW: nahezu 100%; 10%iges KW: 30%) und Ascorbinsäure (ca. 90% bei AS 200 µg/ml) gehemmt. TGF-ß2 stimulierte die Proliferation bis zu einem 3fachen Wert der Kontrollen. Die Migration wurde durch humanes KW (unverdünntes KW: nahezu 100%; 10%iges KW: 30%) und TGF-ß2 (ca. 50% bei allen Konzentrationen) gehemmt. AS hatte einen stimulierenden Einfluß (20-50%) auf die Migration. Schlußfolgerung: Noch ist unklar, welche Substanzen im KW für den proliferationshemmenden Einfluß auf korneale Endothelzellen verantwortlich sind. Sowohl AS als auch TGF-ß2 zeigten einen gegensätzlichen Effekt auf Proliferation und Migration kultivierter BCEC. Die Bedeutung dieser Beobachtungen für die Wundheilung humaner kornealer Endothelzellen in vivo ist Gegenstand weiterer Untersuchungen. / Purpose: Corneal endothelial cells are non-proliferating, wound healing primarily occurs by migration of adjacent cells. Endothelial cells in vivo are constantly exposed to aqueous humor (AH). Elevated concentrations of TGF-ß2 and ascorbic acid (AA) are present in aqueous humor. The influence of human AH, TGF- ß2 and AA on proliferation and migration of bovine corneal endothelial cells in vitro was investigated. Methods: Proliferation assays: BCEC at first passage were seeded at 1.5x104 cells/well. Fresh medium containing human AH (10% and 100%), TGF-ß2 (0,1;1 and 10 ng/ml) and AA (50, 100 and 200 µg/ml) was added to the cells. 72 or 96 hours later the cells were counted. Migration assays: BCEC at first passage were grown to confluency. A central, circular "wound" was made with an especially designed trephine (diameter 5.5 mm). The cultures were incubated with fresh medium containing human AH, TGF-ß2 and AA in the same concentrations for 72 or 96 hours. The cells were then counted in five randomly chosen sections from the wound edge. Results: Proliferation of BCE cells was inhibited by human AH (pure AH: nearly 100%, 10% AH: 30%) and AA (about 90% at 200 µg/ml). TGF-ß2 stimulated the proliferation up to 3 fold compared to the controls. Migration was inhibited by human AH (pure AH: nearly 100%, 10% AH: 30%) and TGF-ß2 (about 50% at all concentrations). AA had a stimulatory effect (20-50%) on migration. Conclusion: Presently, it remains unknown which substances in AH are responsible for the inhibiting effect on corneal endothelial proliferation. Both TGF-ß2 and AA showed to have a differential effect on proliferation and migration of cultured BCE cells. The significance of these observations for wound healing of human corneal endothelial cells in vivo has to be investigated.

Das Ziel dieser Arbeit war die Untersuchung der Rollgeschwindigkeiten von Leukozyten auf humanen kardialen mikrovaskulären Endothelzellen (HCMEC) und humanen umbilikalen venösen Endothelzellen (HUVEC). Die Endothelzellen wurden aus explantierten humanen Herzen sowie aus menschlichen Nabelschnüren unmittelbar postpartal gewonnen. Unter definierten Bedingungen wurden die in einer Flusskammer gemessenen Geschwindigkeiten von L-Selektin exprimierenden Nalm6-IF4 Zellen auf unstimulierten Endothelzellen mit denen auf stimulierten Endothelzellen verglichen. Die langsamere Geschwindigkeit der Leukozyten auf stimulierten Endothelzellen weist darauf hin, dass L-Selektin Liganden auf humanen kardialen mikrovaskulären Endothelzellen erst nach Stimulation exprimiert werden. Die beobachtete Geschwindigkeitsreduktion der Leukozyten ist jedoch von dem in der Literatur beschriebenen Selektin vermittelten Rollen zu unterscheiden. In den Versuchen mit Tunicamycin wurde gezeigt, dass N-glykosidisch gebundene Zucker kritische Bestandteile der Liganden für ihre Interaktion mit L-Selektin sind. Unter den gleichen Versuchsbedingungen wurde auch der Einfluss E-Selektin vermittelter Interaktionen auf die Geschwindigkeit der HL60 Zellen untersucht. Neben dem typischen Rollen wurde hier ebenfalls eine Selektin abhängige Geschwindigkeitsreduktion gesehen. / The aim of the present study was to investigate selectin mediated rolling velocities of leucocytes on human cardiac microvascular endothelial cells (HCMEC) and human umbilical vein endothelial cells (HUVEC). HCMEC were gained from explanted human hearts and HUVEC from umbilical cords immediately postpartum. Flow velocities of L-Selectin expressing Nalm6-IF4 cells on quiesent endothelial cells were compared to those on stimulated endothelial cells. Stimulation of endothelial cells with TNF led to significantly slower velocities of Nalm6-IF4 cells indicating that HCMEC express L-Selectin ligands only after stimulation. The observed reduction of flow velocities differs from rolling of leucocytes described in the literature. Experiments with tunicamycin showed that N-glycosylated carbohydtrate moieties are needed for proper function of L-Selectin ligands. E-Selectin mediated interactions between HL60 cells and endothelial cells were studied under the same conditions. Besides the typical rolling, a selectin mediated reduction of flow velocity was observed.

F-Aktin spielt eine wichtige Rolle bei der Steuerung der endothelialen Barrierefunktion. In dieser Arbeit verwendeten wir Colchicin, Vinca-Alkaloide (Vinblastin, Vincristin) und Paclitaxel um Mikrotubulussysteme (MT) auszulenken und den Effekt auf die Permeabilität zu untersuchen. Endothelzellen wurden auf Polycarbonatfiltermembranen gepflanzt und einem kontinuierlichen hydrostatischen Druck von 10 cm H2O ausgesetzt. Die Exposition von Endothelzell-Monolayern gegenüber Colchicin und Vinca-Alkaloiden führte innerhalb von 60 100 Minuten zeit- und dosisabhängig zu einem fünf zehnfachen Anstieg der hydraulischen Konduktivität. Dagegen war nach MT-Stabilisation durch Paclitaxel keine Permeabilitätszunahme festzustellen. Doppelimmunfluoreszenz-Mikroskopie zeigte, dass die MT-Depolymerisation durch Colchicin und Vinca Alkaloide zu F-Aktin-Umverteilung, Stressfaserbildung und Zellretraktionen mit ausgeprägter parazellulärer Lücken-Bildung führt. Diese Phänomene wurden durch Kombinationen von Vinblastin und Paclitaxel deutlich abgeschwächt. Die fluorometrische Messung des intrazellulären F-Aktins nach MT-Depolymerisation durch Vinblastin resultierte in einer signifikanten Zunahme der Aktinfilamente. Auf der anderen Seite resultierte F-Aktin Abbau durch Cytochalasin D und Clostridium difficile (TcdB-10463) morphologisch nicht in einer Veränderung von MT-Strukturen. Dabei zeigten in Interzellularbrücken gelegene MT-Filamente Kolokalisation mit F-Aktin Fragmenten. Unsere Ergebnisse demonstrieren, dass MT-Systeme an der Regulation der endothelialen Barriere beteiligt sind. Darüber hinaus verdeutlichen die Resultate eine enge Bindung von MT- und Aktin-Filamenten innerhalb endothelzellulärer Adhäsionskontakte. / The endothelian cytosceleton plays an important role in the regulation of endothelial permeability via cellular actin filaments. We tested the effect of agents known to perturb cellular microtubules on the permeability of endothelial cell monolayers. The agents chosen were colchicine, the vinca alkaloids vinblastine and vincristine and paclitaxel. Cell monolayers were prepared on polycarbonate filter membranes and exposed to a continuous hydrostatic pressure of 10 cm H2O. Colchicine and the vinca alkaloids caused a five to tenfold increase in the hydraulic conductivity of the monolayers within 60 100 min. The effect was dose and time dependent. The microtubule stabilizer paclitaxel caused no increase in permeability. Double-immunofluorescence microscopy showed that microtubule depolymerisation was associated with certain morphological features such as inter-endothelial gaps, cell retraction, f-actin reorganisation and some stressfibre appearance. These phenomena were significantly reduced when vinblastine and paclitaxel were combined. Measurement of intracellular f-actin following microtubule inhibition with vinblastine showed a significant increase in endothelial actin filaments. No changes in microtubule structures were seen when actin filaments were perturbed with cytochalasin D and Clostridium difficile (TcdB-10463). However, in this case the intercellular bridges showed that microtubules were co-localised with fragments of actin filaments from neighbouring cells. Our data demonstrate that microtubules are important for the regulation of endothelial permeability. Moreover, our results support evidens of binding between microtubules and actin filaments within endothelial cell adhesion contacts.

Während die zellulären Mechanismen der Pathogenese der Arteriosklerose intensiv untersucht worden sind, ist über die Mechanismen, die zu einer bevorzugten Lokalisation arteriosklerotischer Läsionen in bestimmten Gefäßarealen führen, weniger bekannt. Zur Untersuchung dieser Mechanismen wurden Endothelzellen aus menschlichen Koronararterien, einem Gefäßbereich, in dem häufig arteriosklerotische Läsionen beobachtete werden, isoliert und kultiviert. Endothelzellen der Mikrozirkulation menschlicher Herzen wurden unter gleichen Bedingungen kultiviert und die Reaktionen beider Zellarten verglichen. Inkubation der Zellen mit den in Bezug auf die Bildung arteriosklerotischer Plaques besonders pathogenen oxidierten LDL induzierte in makrovaskulären koronaren Endothelzellen eine stärkere Zunahme der PAI-1 Aktivität (182%, p / While the cellular mechanisms of atherosclerosis have been intensively studied, the mechanisms leading to preferential localization of atherosclerotic lesions are less well understood. To further define these mechanisms, endothelial cells from coronary arteries, i.e. vessels with frequent atherosclerotic lesions, were isolated and grown in vitro. In order to compare the reactions of both cell types, endothelial cells derived from microvessels of human hearts were isolated and cultured under identical conditions. Incubation of endothelial cells with oxidized LDL (75 µg/ml protein) induced a significant increase in PAI-1 activity (182 %, p

1981 entwickelten Gerd Binnig und Heinrich Rohrer bei IBM in Zürich das "Scanning Tunneling Microscope". Damit wurde erstmalig das lokal hochaufgelöste Erfassen (bis in den atomaren Auflösungsbereich) von Objekteigenschaften im Nahfeld inerter Oberflächen möglich. Dies und insbesondere die Weiterentwicklung der Technologie und die spätere (1986) Etablierung der Rasterkraftmikroskopie (Atomic Force Microscopy - AFM), die diese Auflösungsmöglichkeiten der Rastersondenmikroskope auch an Non-Konduktoren (nicht leitende Untersuchungsoberflächen) realisieren konnte, stellte die Geburtsstunde einer neuen mikroskopischen Ära auf dem Gebiet der biomedizinischen Grundlagenforschung dar (Kapitel 1.3). Das Studium der umfangreichen Literaturquellen zu diesem Thema und der direkte wissenschaftliche Kontakt und Erfahrungsaustausch mit anderen AFM- Arbeitsgruppen ließen im Initialstadium dieser vorliegenden Arbeit bereits erkennen, dass in der kardiovaskulären Grundlagenforschung zunehmend rasterkraftmikroskopische Versuchsansätze bearbeitet und kardiologisch interessante Fragestellungen mittels dieser Methode begleitend untersucht wurden (Kapitel 1.4). Das Ziel dieser vorliegenden Arbeit bestand darin, kardiovaskulär relevante Zellen und Einzelproteine in vivo und interventionelle Materialien (Stents) rasterkraftmikroskopisch zu untersuchen, wobei die Etablierung und technisch aufwendige Optimierung dieser neuen mikroskopischen (Kapitel 3.1) und der zellspezifisch präparatorischen Methoden (Kapitel 3.2) an diesen Untersuchungsobjekten im Mittelpunkt stehen sollte. Die im Rahmen dieser Arbeit untersuchten endothelialen Zellen und H9C2-Myozyten stammten aus, in unserem Forschungslabor etablierten, immortalen Kulturzelllinien. Die adulten und Kardiomyozyten neonataler Ratten, die kardial- fibrozytären Zellen sowie die Thrombozyten wurden primär isoliert und als Primärkulturzellen kultiviert (Kapitel 3.2.3 und 3.2.4). Außerdem wurden vitale aortale Endothelzellen unterschiedlicher Tiere (Ratte, Meerschwein, Kaninchen) im Gewebsverband der thorakalen Aorta untersucht (Kapitel 4.2). Die Zellen wurden initial, im Rahmen der Etablierungsphase mittels unterschiedlicher Methoden fixiert und nachfolgend rasterkraftmikroskopisch untersucht und dargestellt. Der Etablierungsprozess der Methodik begann mit der Abbildung luftgetrockneter Zellen (Kapitel 4.1.1) unter Raumbedingungen und setzte sich über verschiedene Modifikationen der Zellpräparation (z.B. Glutardialdehydfixation, Cryofixation), des Abbildungsmodus (Contact-, Non-Contact-, Tapping-Mode) und der Abbildungsbedingungen (Raumbedingungen, zellphysiologische Umgebung) fort, so dass schließlich die Abbildung vitaler Zellen (Kapitel 4.1.2 und Kapitel 4.2 - 4.5) in ihrer strukturellen und funktionellen Umgebung (z.B. aortale Endothelzellen im Gewebsverband) etabliert werden konnte und routinemäßig reproduzierbar war. An stabilen oder künstlich stabilisierten Strukturen der o.g. vitalen Zellen wurden erste orientierende Messungen der bioelastischen Eigenschaften (Kraft-Abstands-Kurven, Kapitel 4.1.2.1) durchgeführt. Außerdem haben wir im Einzelfall, wenn technisch und apparativ möglich, andere hochauflösende strukturanalytische Verfahren (z.B. TEM) als mikroskopische Referenzuntersuchungen herangezogen (Kapitel 4.1.2; 4.4.1; 4.6), wobei z.T. erstaunliche Übereinstimmung zwischen den AFM- Daten und den strukturanalytischen Daten der Referenzmethoden nachweisbar waren. Ein strukturell durch Elektronenmikroskopie und Röntgendiffraktionsanalyse sehr gut beschriebenes komplexes Funktionsprotein, das 20-S-Proteasom, wurde mittels der Rasterkraftmikroskopie abgebildet und vermessen und die so gewonnenen strukturanalytischen Daten mit den bekannten strukturellen Abmessungen des Proteins verglichen (Kapitel 4.6). Die hierbei detektierten dimensionalen Abweichungen zwischen den AFM- assoziierten Daten und den bekannten strukturanalytischen Daten der Elektronenmikroskopie wurden im Kontext der funktionellen Integrität des Proteins und hinsichtlich möglicher methodischer Fehlereinflüsse (Kapitel 3.1.4.3) diskutiert. Interventionelle Materialien (Stents), die in der täglichen kardiologischen Praxis Anwendung finden, sind hinsichtlich ihrer Ultrastruktur mittels dieser hochsensitiven Abbildungsmethode im Nahfeld von Objektoberflächen untersucht worden. Bezüglich ihrer nativen Oberflächenbeschaffenheit und ihrer mechanischen Alteration durch den Ballon- Dilatationsprozess wurden die Stents sehr detailliert qualitativ und quantitativ (Kapitel 4.7) beschrieben, wobei Prädilektionsstellen der prozedural- assoziierten mechanischen Beanspruchung der Stents durch die hier beschriebene, oberflächensensitive AFM- Methode sehr genau diskriminiert werden konnten. Die präparierten Stents wurden weiterführend mit humanen Thrombozytenkonzentraten inkubiert und die Zell- Stentoberflächenkontakte sowie mögliche Stentoberflächen- induzierte Veränderungen der Thrombozyten sind morphologisch ausführlich beschrieben worden. Letztendlich wurde im Rahmen der vorliegenden Arbeit die spezifische Aktivierung der vitalen Thrombozyten durch pharmakologische Stimulantien (z.B. ADP) mit der, durch den AFM-Abbildungsprozess induzierten Thrombozytenaktivierung (Kapitel 4.5) unter AFM-Bedingungen verglichen und diskutiert. Die Ergebnisse dieser Arbeit weisen, dass mit der AFM-Technologie und objektorientiert optimierten Mess- und Präparationsmethoden ein neues mikroskopisches Analyseverfahren vorliegt, dass zum einen real-dreidimensionale morphologische Bildgebung bis in den submolekularen Auflösungsbereich an vitalen Zellen und präparierten Proteinkomplexen, zum anderen aber gleichermaßen Funktionsanalytik in Form von Messungen zelldynamischer Prozesse wie Migrationsbewegungen und Kontraktionen sowie visko- elastische Quantifizierung von Zellmembranen erlaubt. Der Vorteil gegenüber den meisten gegenwärtig verfügbaren mikroskopischen Methoden liegt in der neu eröffneten Möglichkeit der seriellen, wiederholten und stabil reproduzierbaren Messung an vitalen Zellen und zellulären Substrukturen. Insofern könnte in Zukunft diese neue Technologie eine methodische Bereicherung der mikroskopisch-morphologisch und funktionell orientierten Analysetechnik darstellen. / In 1981 Binnig and Rohrer invented the "Scanning Tunneling Microscope". Thereby it became feasible to high-resolution record the surface-properties of specimens (up to atomic resolution) at the nearfield of inert surfaces. This and in detail the further development of this technology and the establishment of "Atomic Force Microscopy" (1986), that allows implementation of this resolution capabilities in non-conductors or insulating materials represent the birth of a new microscopic era in the field of biomedical basic research (chapter 1.3). The promise of atomic (scanning) force microscopy (AFM) for cardiovascular research is enormous. The perusal of the extensive literature concerning this topic and scientific contact with other researchers reveals initial the capabilities of this method in cardiovascular basic research. Intriguing questions of cardiology may investigate concomitantly with help of scanning-force-micoscopic approaches (chapter 1.4). The aim of this study was to investigate relevant cardiovascular cells and single proteins in-vivo and specific materials (coronary artery stents) with scanning-force-micoscopic setup. The establishment and expensive optimization of this new microscopic method (chapter 3.1) and of the cell specific preparatory methods (chapter 3.2) represented the center of interest of our inevestigations. The endothelial cells and H9C2-myocytes stem from established imortal cell culture lines. The adult cardiomyocytes and cardiomyocytes of neonatal rats, the fibrocytes and the thrombocytes were primarily cultivated (chapter 3.2.3 and 3.2.4). In addition we investigated aortic endothelial cells of intact aortic tissue of different animals (rat, guinea pig, rabbit - chapter 4.2). During the establish experiments cells underlied different methods of cell-fixation. The primary investigations was performed using air-dried cells (chapter 4.1.1) analyzed in room ambient conditions and were continued by different modifications of cell-preparation. (e.g. glutardialdehyde-fixation, cryo-fixation), of microscopic mode (contact-, non-contact-, tapping-mode) and of cell-specific environmental conditions (from room ambient to cellphysiological medium and temperature). As result we became enabled to investigate (reproducible and routinely) vital cells (chapter 4.1.2 and chapter 4.2 - 4.5) embedded in physiological normal structural und functional ambient conditions (e.g. endothelial cells of intact aortic tisue in-vivo). Additionally, we performed measurements of bio-elastic properties of stable or artificial stabilized structures of named cells (force-distances-curves - chapter 4.1.2.1). If posibble, depending of available technical equipment, we compared our microscopic results with other high-resolution analytical procedures of reference (e.g. TEM - Kapitel 4.1.2; 4.4.1; 4.6) and detected astonishing congruence between the data. Furthermore we analyzed the well-described (electron-microscopy and x-ray-diffraction data) complex 20-S-proteasome using a specific atomic force microscopic setup. Analytical and structural data of these AFM-scans and abovementioned methods were likened (chapter 4.6). The deviations concerning the detected proportions were discussed regarding functional integrity of the protein and with respect to potential methodically determined artifacts. (chapter 3.1.4.3). Assaying (qualitative and quantitative) the surface roughness properties of coronary artery stents, we found significant alterations of stent material induced by balloondilatation. We suppose, that changes in roughness of inner surface of coronary artery stents might induce clinical problems like acute stent-thrombosis and in-stent-restenosis. Finally these stents were coated with human thromboytes to investigate cell-stent-surface interactions. Surface-roughness correllated triggering of thrombocyte adhesion was evaluated by morphological analysis of AFM-scans. Finishing, we have investigated and concluding discussed the specific activation of vital thrombocytes by pharmacological substances (e.g. ADP) and by mechanical stimulation (due to AFM-associated tip-surface-interaction). The results of this work demonstrate, AFM-technology using optimized microscopic setup and object-specific adjusted measurement- and preparation- methods, is an new, powerful, microscopic technique, that allow real-3-dimensional morphological mapping up to submolecular range of resolution in vital cells and protein complexes. Moreover, this technology opens new dimensions in functional analytic of cell migration processes or cellular contractions and in evaluation of visco-elastic quantification of cell membranes. The advantage owed to the most currently available microscopic methods is the option of serial and reproducible measurement of vital cells and subcellular structures. In this respect, this new method might represent a methodical enrichment of the microscopic-morphological and functional oriented analysis-technique in future.

No description available.

No description available.

Bone fractures and pathologic conditions like chronic wounds significantly reduce the quality of life for the patients, which is especially dramatic in an elderly population with considerable multi-morbidity and lead to substantial socio-economic costs. To improve the wound healing capacity of these patients, new strategies for the design of novel multi-functional biomaterials are required: they should be able to decrease extensive pathologic tissue degradation and specifically control angiogenesis in damaged vascularized tissues like bone and skin. Glycosaminoglycans (GAGs) like hyaluronan (HA) and chondroitin sulfate (CS) as important extracellular matrix (ECM) components are involved in several biological processes such as matrix remodeling and growth factor signaling, either by directly influencing the cellular response or by interacting with mediator proteins. This could be useful in functionalizing biomaterials, but native sulfated GAGs (sGAGs) show a high batch-to-batch variability and are limited in their availability. Chemically modified HA and CS derivatives with much more defined characteristics regarding their carbohydrate backbone, sulfate group distribution and sulfation degree are favorable to study the structure-function relationship of GAGs in their interaction with mediator proteins and/or cells and this might be used to precisely modulate activity profiles to stimulate wound healing. By combining collagen type I as the main structural protein of the bone and skin ECM with these GAG derivatives, 2.5-dimensional (2.5D) and 3D artificial ECM (aECM) coatings and hydrogels were developed. These biomaterials as well as the respective GAG derivatives alone were compared to native GAGs and used to analyze how the sulfation degree, pattern and carbohydrate backbone of GAGs influence: i) the activity of tissue inhibitor of metalloproteinase-3 (TIMP-3) and vascular endothelial growth factor-A (VEGF-A) as main regulators of ECM remodeling and angiogenesis, ii) the composition and characteristics of the developed 2.5D and 3D aECMs, iii) the enzymatic degradation of collagen-based aECMs and HA/collagen-based hydrogels, iv) the proliferation and functional morphology of endothelial cells. Surface plasmon resonance (SPR) and enzyme linked immunosorbent assay (ELISA) binding studies revealed that sulfated HA (sHA) derivatives interact with TIMP-3 and VEGF-A in a sulfation-dependent manner. sHA showed an enhanced interplay with these proteins compared to native GAGs like heparin (HEP) or CS, suggesting a further impact of the carbohydrate backbone and sulfation pattern. sGAGs alone were weak modulators of the matrix metalloproteinase-1 and -2 (MMP-1 and -2) activity and did not interfere with the inhibitory potential of TIMP-3 against these proteinases during enzyme kinetic analyses. However, the formation of TIMP 3/GAG complexes reduced the binding of TIMP-3 to cluster II and IV of its endocytic receptor low-density lipoprotein receptor-related protein-1 (LRP-1, mediates the up-take and degradation of TIMP-3 from the extracellular environment) in a sulfation- and GAG type-dependent manner. It is of note that the determined complex stabilities of TIMP-3 with cluster II and IV were almost identical indicating for the first time that both clusters contribute to the TIMP-3 binding. Competitive SPR experiments demonstrated that GAG polysaccharides interfere stronger with the TIMP 3/LRP-1 interplay than GAG oligosaccharides. The importance of the position of sulfation is highlighted by the finding that a sHA tetrasaccharide exclusively sulfated at the C6 position of the N-acetylglucosamine residues significantly blocked the receptor binding, while CS and HEP hexasaccharides had no detectable effects. Thus, sHA derivatives as part of biomaterials could be used to sequester and accumulate TIMP 3 in aECMs in a defined manner where sHA-bound TIMP-3 could decrease the matrix breakdown by potentially restoring the MMP/TIMP balance. GAG binding might extend the beneficial presence of TIMP-3 into wounds characterized by excessive pathologic tissue degradation (e.g. chronic wounds, osteoarthritis). Mediator protein interaction studies with sHA coated surfaces showed the simultaneous binding of TIMP-3 and VEGF-A, even though the sHA/VEGF-A interplay was preferred. Moreover, kinetic analysis revealed almost comparable affinities of both proteins for VEGF receptor-2 (VEGFR-2), explaining their competition that mainly regulates the activation of endothelial cells. Additional SPR measurements demonstrated that the binding of sGAGs to TIMP-3 or VEGF-A decreases the binding of the respective mediator protein to VEGFR-2. Likewise, a sulfation-dependent reduction of the binding signal was observed after pre-incubation of a mixture of TIMP-3 and VEGF-A with sGAG poly- and oligosaccharides. The biological consequences of GAGs interfering with VEGF-A/VEGFR-2 and TIMP-3/VEGFR 2 were assessed in vitro using porcine aortic endothelial cells stably transfected with VEGFR 2 (PAE/KDR cells). The presence of sHA both decreased VEGF-A activity and the activity of TIMP-3 to inhibit the VEGF-A-induced VEGFR-2 phosphorylation. The same decreased activities could be observed for the migration of endothelial cells. However, if sHA, TIMP-3 and VEGF-A were present simultaneously, sHA partially restored the TIMP-3-mediated blocking of VEGF-A activity. These findings provide novel insights into the regulatory potential of sHA during endothelial cell activation as an important aspect of angiogenesis, which could be translated into the design of biomaterials to treat abnormal angiogenesis. These sHA-containing materials might control the angiogenic response by modulating the activity of TIMP 3 and VEGF-A. The in vitro fibrillogenesis of collagen type I in the presence of sHA derivatives led to 2.5D collagen-based aECM coatings with stable collagen contents and GAG contents that resemble the organic part of the bone ECM. A burst release of GAGs was observed during the first hour of incubation in buffer with the GAG content remaining almost constant afterwards, implying that the number of GAG-binding sites of collagen restricts the amounts of associated GAGs. Moreover, two differently sulfated HA derivatives could for the first time be incorporated into one multi-GAG aECM as verified via agarose gel electrophoresis and fluorescence measurements. This illustrates the multiple options to modify the aECM composition and thereby potentially their functionality. Atomic force microscopy showed that the presence of sHA derivatives during fibrillogenesis significantly reduced the resulting fibril diameter in a concentration- and sulfation-dependent manner, indicating an interference of the GAGs with the self-assembly of collagen monomers. In line with enzyme kinetic results, none of the GAGs as part of aECMs altered the enzymatic collagen degradation via a bacterial collagenase. Thus aECMs were proven to be biodegradable independent from their composition, which is favorable concerning a potential biomedical usage of the aECMs e.g. as implant coatings. HA/collagen-based hydrogels containing fibrillar collagen embedded into a network of crosslinked HA and sGAGs were developed as 3D aECMs. Scanning electron microscopy demonstrated a porous structure of the gels after lyophilization, which could favor the cultivation of cells. The presence of collagen markedly enhanced the stability of the gels against the enzymatic degradation via hyaluronidase, something beneficial to clinical use as this is often limited by the generally fast breakdown of HA. Binding and release experiments with lysozyme, as positively charged model protein for e.g. pro-inflammatory cytokines, and VEGF A revealed that the sulfation of GAGs increased the protein binding capacity for pure GAG coatings and retarded the protein release from hydrogels compared to hydrogels without sGAGs. Moreover, the additional acrylation of sHA was shown to strongly reduce the interaction with both proteins when the primary hydroxyl groups were targets of acrylation. This stresses the influence of the substitution pattern on the protein binding properties of the GAG derivatives. However, hydrogel characteristics like the elastic modulus remained unaffected. The different interaction profiles of lysozyme and VEGF-A with GAGs demonstrated a protein-specific preference of different monosaccharide compositions, suggesting that the mediator protein binding could be simultaneously adjusted for several proteins by combining different GAG derivatives. This might allow the scavenging of pro-inflammatory cytokines and at the same time a binding and release of wound healing stimulating growth factors. Since there is a growing demand for biomaterials to regenerate injured vascularized tissues like bone and skin, endothelial cells were used to examine the direct effects of solute GAGs and hydrogels containing these GAGs in vitro. In both cases, sHA strongly enhanced the proliferation of PAE/KDR cells. A VEGFR-2-mediated effect of GAGs on endothelial cells as underlying mechanism is unlikely since GAGs alone did not bind to VEGFR-2 and had no influence on VEGFR-2 phosphorylation. Other factors like GAG-induced alterations of cell-matrix interactions and cell signaling could be responsible. In accordance with SPR results, a decreased endothelial cell proliferation stimulating activity of VEGF-A was observed in the presence of solute GAGs or after binding to hydrogels compared to the respective treatment without VEGF-A. However, tube formation could be observed in the presence of solute VEGF A and GAGs and within hydrogels with sGAGs that released sufficient VEGF-A amounts over time. Overall the presence of GAGs and VEGF-A strongly promoted the endothelial cell proliferation compared to the treatment with GAGs or VEGF-A alone. Thus, HA/collagen-based hydrogels functionalized with sHA derivatives offer a promising option for the design of “intelligent” biomaterials that direct and regulate the cellular behavior instead of simply acting as inert filling material. They could be used for the controlled delivery and/or scavenging of multiple mediator proteins, thus enhancing the local availability or reducing the activity of these GAG-interacting mediator proteins, or by directly influencing the cellular response. This might be applied to a range of pathological conditions by tuning the biomaterial compositions to patient-specific needs. However, extensive in vivo validation is required to show whether these in vitro findings could be used to control the biological activity of for instance TIMP-3 and VEGF-A, especially under the pathological conditions of extended matrix degradation and dysregulated angiogenesis.

Bone fractures and pathologic conditions like chronic wounds significantly reduce the quality of life for the patients, which is especially dramatic in an elderly population with considerable multi-morbidity and lead to substantial socio-economic costs. To improve the wound healing capacity of these patients, new strategies for the design of novel multi-functional biomaterials are required: they should be able to decrease extensive pathologic tissue degradation and specifically control angiogenesis in damaged vascularized tissues like bone and skin. Glycosaminoglycans (GAGs) like hyaluronan (HA) and chondroitin sulfate (CS) as important extracellular matrix (ECM) components are involved in several biological processes such as matrix remodeling and growth factor signaling, either by directly influencing the cellular response or by interacting with mediator proteins. This could be useful in functionalizing biomaterials, but native sulfated GAGs (sGAGs) show a high batch-to-batch variability and are limited in their availability. Chemically modified HA and CS derivatives with much more defined characteristics regarding their carbohydrate backbone, sulfate group distribution and sulfation degree are favorable to study the structure-function relationship of GAGs in their interaction with mediator proteins and/or cells and this might be used to precisely modulate activity profiles to stimulate wound healing. By combining collagen type I as the main structural protein of the bone and skin ECM with these GAG derivatives, 2.5-dimensional (2.5D) and 3D artificial ECM (aECM) coatings and hydrogels were developed. These biomaterials as well as the respective GAG derivatives alone were compared to native GAGs and used to analyze how the sulfation degree, pattern and carbohydrate backbone of GAGs influence: i) the activity of tissue inhibitor of metalloproteinase-3 (TIMP-3) and vascular endothelial growth factor-A (VEGF-A) as main regulators of ECM remodeling and angiogenesis, ii) the composition and characteristics of the developed 2.5D and 3D aECMs, iii) the enzymatic degradation of collagen-based aECMs and HA/collagen-based hydrogels, iv) the proliferation and functional morphology of endothelial cells. Surface plasmon resonance (SPR) and enzyme linked immunosorbent assay (ELISA) binding studies revealed that sulfated HA (sHA) derivatives interact with TIMP-3 and VEGF-A in a sulfation-dependent manner. sHA showed an enhanced interplay with these proteins compared to native GAGs like heparin (HEP) or CS, suggesting a further impact of the carbohydrate backbone and sulfation pattern. sGAGs alone were weak modulators of the matrix metalloproteinase-1 and -2 (MMP-1 and -2) activity and did not interfere with the inhibitory potential of TIMP-3 against these proteinases during enzyme kinetic analyses. However, the formation of TIMP 3/GAG complexes reduced the binding of TIMP-3 to cluster II and IV of its endocytic receptor low-density lipoprotein receptor-related protein-1 (LRP-1, mediates the up-take and degradation of TIMP-3 from the extracellular environment) in a sulfation- and GAG type-dependent manner. It is of note that the determined complex stabilities of TIMP-3 with cluster II and IV were almost identical indicating for the first time that both clusters contribute to the TIMP-3 binding. Competitive SPR experiments demonstrated that GAG polysaccharides interfere stronger with the TIMP 3/LRP-1 interplay than GAG oligosaccharides. The importance of the position of sulfation is highlighted by the finding that a sHA tetrasaccharide exclusively sulfated at the C6 position of the N-acetylglucosamine residues significantly blocked the receptor binding, while CS and HEP hexasaccharides had no detectable effects. Thus, sHA derivatives as part of biomaterials could be used to sequester and accumulate TIMP 3 in aECMs in a defined manner where sHA-bound TIMP-3 could decrease the matrix breakdown by potentially restoring the MMP/TIMP balance. GAG binding might extend the beneficial presence of TIMP-3 into wounds characterized by excessive pathologic tissue degradation (e.g. chronic wounds, osteoarthritis). Mediator protein interaction studies with sHA coated surfaces showed the simultaneous binding of TIMP-3 and VEGF-A, even though the sHA/VEGF-A interplay was preferred. Moreover, kinetic analysis revealed almost comparable affinities of both proteins for VEGF receptor-2 (VEGFR-2), explaining their competition that mainly regulates the activation of endothelial cells. Additional SPR measurements demonstrated that the binding of sGAGs to TIMP-3 or VEGF-A decreases the binding of the respective mediator protein to VEGFR-2. Likewise, a sulfation-dependent reduction of the binding signal was observed after pre-incubation of a mixture of TIMP-3 and VEGF-A with sGAG poly- and oligosaccharides. The biological consequences of GAGs interfering with VEGF-A/VEGFR-2 and TIMP-3/VEGFR 2 were assessed in vitro using porcine aortic endothelial cells stably transfected with VEGFR 2 (PAE/KDR cells). The presence of sHA both decreased VEGF-A activity and the activity of TIMP-3 to inhibit the VEGF-A-induced VEGFR-2 phosphorylation. The same decreased activities could be observed for the migration of endothelial cells. However, if sHA, TIMP-3 and VEGF-A were present simultaneously, sHA partially restored the TIMP-3-mediated blocking of VEGF-A activity. These findings provide novel insights into the regulatory potential of sHA during endothelial cell activation as an important aspect of angiogenesis, which could be translated into the design of biomaterials to treat abnormal angiogenesis. These sHA-containing materials might control the angiogenic response by modulating the activity of TIMP 3 and VEGF-A. The in vitro fibrillogenesis of collagen type I in the presence of sHA derivatives led to 2.5D collagen-based aECM coatings with stable collagen contents and GAG contents that resemble the organic part of the bone ECM. A burst release of GAGs was observed during the first hour of incubation in buffer with the GAG content remaining almost constant afterwards, implying that the number of GAG-binding sites of collagen restricts the amounts of associated GAGs. Moreover, two differently sulfated HA derivatives could for the first time be incorporated into one multi-GAG aECM as verified via agarose gel electrophoresis and fluorescence measurements. This illustrates the multiple options to modify the aECM composition and thereby potentially their functionality. Atomic force microscopy showed that the presence of sHA derivatives during fibrillogenesis significantly reduced the resulting fibril diameter in a concentration- and sulfation-dependent manner, indicating an interference of the GAGs with the self-assembly of collagen monomers. In line with enzyme kinetic results, none of the GAGs as part of aECMs altered the enzymatic collagen degradation via a bacterial collagenase. Thus aECMs were proven to be biodegradable independent from their composition, which is favorable concerning a potential biomedical usage of the aECMs e.g. as implant coatings. HA/collagen-based hydrogels containing fibrillar collagen embedded into a network of crosslinked HA and sGAGs were developed as 3D aECMs. Scanning electron microscopy demonstrated a porous structure of the gels after lyophilization, which could favor the cultivation of cells. The presence of collagen markedly enhanced the stability of the gels against the enzymatic degradation via hyaluronidase, something beneficial to clinical use as this is often limited by the generally fast breakdown of HA. Binding and release experiments with lysozyme, as positively charged model protein for e.g. pro-inflammatory cytokines, and VEGF A revealed that the sulfation of GAGs increased the protein binding capacity for pure GAG coatings and retarded the protein release from hydrogels compared to hydrogels without sGAGs. Moreover, the additional acrylation of sHA was shown to strongly reduce the interaction with both proteins when the primary hydroxyl groups were targets of acrylation. This stresses the influence of the substitution pattern on the protein binding properties of the GAG derivatives. However, hydrogel characteristics like the elastic modulus remained unaffected. The different interaction profiles of lysozyme and VEGF-A with GAGs demonstrated a protein-specific preference of different monosaccharide compositions, suggesting that the mediator protein binding could be simultaneously adjusted for several proteins by combining different GAG derivatives. This might allow the scavenging of pro-inflammatory cytokines and at the same time a binding and release of wound healing stimulating growth factors. Since there is a growing demand for biomaterials to regenerate injured vascularized tissues like bone and skin, endothelial cells were used to examine the direct effects of solute GAGs and hydrogels containing these GAGs in vitro. In both cases, sHA strongly enhanced the proliferation of PAE/KDR cells. A VEGFR-2-mediated effect of GAGs on endothelial cells as underlying mechanism is unlikely since GAGs alone did not bind to VEGFR-2 and had no influence on VEGFR-2 phosphorylation. Other factors like GAG-induced alterations of cell-matrix interactions and cell signaling could be responsible. In accordance with SPR results, a decreased endothelial cell proliferation stimulating activity of VEGF-A was observed in the presence of solute GAGs or after binding to hydrogels compared to the respective treatment without VEGF-A. However, tube formation could be observed in the presence of solute VEGF A and GAGs and within hydrogels with sGAGs that released sufficient VEGF-A amounts over time. Overall the presence of GAGs and VEGF-A strongly promoted the endothelial cell proliferation compared to the treatment with GAGs or VEGF-A alone. Thus, HA/collagen-based hydrogels functionalized with sHA derivatives offer a promising option for the design of “intelligent” biomaterials that direct and regulate the cellular behavior instead of simply acting as inert filling material. They could be used for the controlled delivery and/or scavenging of multiple mediator proteins, thus enhancing the local availability or reducing the activity of these GAG-interacting mediator proteins, or by directly influencing the cellular response. This might be applied to a range of pathological conditions by tuning the biomaterial compositions to patient-specific needs. However, extensive in vivo validation is required to show whether these in vitro findings could be used to control the biological activity of for instance TIMP-3 and VEGF-A, especially under the pathological conditions of extended matrix degradation and dysregulated angiogenesis.