Le remodelage bronchique regroupe des entités physiopaphologiques commel’hypertrophie musculaire lisse dans l’asthme ou l’augmentation d’épaisseur bronchique surl’infiltration de cellules inflammatoires et l’accumulation de fibrose dans la BPCO. Cesremodelages sont corrélés à l’obstruction fonctionnelle et donc à la sévèrité de ces maladies.L’analyse de biopsies bronchique ou pulmonaire permet d’étudier ce phénoméne qui, aprèsune meilleure compréhension, est une cible thérapeutique intérressante. Le premier articleest une revue d’indications de bronchoscopie chez les patients de réanimation. La deuxièmeétude a montré une augmentation des fibrocytes sanguins au cours d’exacerbation sévèrede patient BPCO et une corrélation entre leur taux et le risque de décès du patient. La voiede signalisation du CXCR4 semble impliquée dans ce recrutement. La troisième étudecherche à explorer la localisation et les caractéristiques intra-pulmonaires des fibrocyteschez le patient BPCO à l’état stable. La quatrième étude a montré, in vivo, que le gallopamil,un inhibiteur calcique, pouvait diminuer la taille de muscle lisse bronchique de patientasthmatique sévère en ciblant la biogenèse mitochondriale. Ceci pourrait en faire une armethérapeutique interressante et totalement novatrice. La dernière étude a permis d’isoler unphénotype de patient asthmatique non sévère à « muscle lisse bronchique augmenté » quiprésente un risque accru d’exacerbation et de contrôle non optimal de leur asthme. Lesmitochondries semblent jouer un rôle clé comme dans l’asthme sévère. / Airway remodeling groups pathophysiological entities such as smooth musclehypertrophy in asthma or increase bronchial thickness due to infiltration of inflammatory cellsand fibrosis in COPD. These remodeling is correlated with the functional obstruction andtherefore with the severity of these diseases. The bronchial or lung biopsies analysis allowsto study this phenomenon which, after understanding, is an interesting therapeutic target.The first article is a review of indications of bronchoscopy in critically ill patients. The secondstudy showed an increase in blood fibrocytes during severe exacerbation of COPD patientand a correlation between their rate and the risk of patient death. CXCR4 signaling pathwayseems to be involved in the fibrocyte recruitment. The third study seeks to explore thelocation and characteristics of intra-pulmonary fibrocytes in stable COPD patients. The fourthstudy has shown, in vivo, that gallopamil, a calcium channel blocker, could reduce airwaysmooth muscle size in severe asthmatic patient by targeting mitochondrial biogenesis. Thiscould make it an interesting therapeutic weapon and totally innovative. The last study hasisolated a non-severe asthma phenotype with "increased bronchial smooth muscle," whichpresents an increased risk of exacerbation and a suboptimal control of their asthma. Themitochondria appear to play a key role as in severe asthma.

Immune cells such as neutrophils and monocytes enter tissues after tissue damage and clear cell debris to allow repair cells such as fibroblasts to close the wound. Monocytes also differentiate into fibroblast-like cells called fibrocytes to mediate wound healing, similar to fibroblasts. However, in abnormal wound healing such as acute respiratory distress syndrome (ARDS) and fibrosing diseases, the accumulation of immune cells such as neutrophils or fibrocytes become detrimental to health. In ARDS, neutrophils accumulate in the lungs and causes additional damage by producing reactive oxygen species (ROS). In fibrosing diseases, increased fibrocyte differentiation is one of the causes that increase extracellular matrix deposition, which leads to severe scar tissue build up. Since there are no effective treatments for ARDS or fibrosing diseases, understanding the regulation of neutrophil activation or fibrocyte differentiation could be helpful to develop new effective therapies. The Gomer lab has found several factors that either promote or inhibit fibrocyte differentiation. The pro-fibrotic cytokines such as IL-4 and IL-13 potentiate fibrocyte differentiation while the plasma protein serum amyloid P (SAP), crosslinked IgG, and the pro-inflammatory cytokines IFN-γ and IL-12 inhibit fibrocyte differentiation. In this thesis, I have now shown that additional factors such as toll-like receptor 2 (TLR2) agonists and low molecular weight hyaluronic acid (LMWHA) inhibit fibrocyte differentiation, while high molecular weight hyaluronic acid (HMWHA) potentiate fibrocyte differentiation. The accumulation of neutrophils in the lungs is one of the major factors that debilitate the health of a patient in ARDS. Since neutrophils have Fc receptors, I examined the effect of SAP on neutrophil spreading, adherence, activation, and accumulation. SAP inhibits neutrophil spreading induced by cell debris and TNF-α induced adhesion, but SAP is unable to have any effect on classic neutrophil adhesion molecules or the production of hydrogen peroxide. SAP inhibits neutrophil accumulation in the lungs of bleomycin-injured mice. There is an exciting possibility of using SAP as a therapeutic agent to treat ARDS.

Os fibrócitos foram inicialmente identificados pelo seu recrutamento rápido para os tecidos lesionados e por atuar diretamente na resposta imune através do reconhecimento, apresentação antigênica e produção de citocinas e quimiocinas. Segundo Grab (2004) fibrócitos podem participar da resposta imune na leishmaniaose e por isso no presente estudo propomos analisar a resposta celular e apresentação antigênica dos fibrócitos na infecção por L. (L.) amazonensis. Para os experimentos in vivo camundongos C57BL/6 e knockout para o receptor TLR-2 foram inoculados na derme auricular com 105 formas promastigotas de L. (L.) amazonensis e 1, 7, 15 dias após a infecção as regiões de inóculo e os linfonodos foram retirados e processados para citometria de fluxo. Para os experimentos in vitro fibrócitos foram obtidos de mononucleares do sangue periférico de camundongos C57BL/6. Os fibrócitos foram avaliados quanto às suas características morfológicas e fenotípicas, infecção, expressão de MHC-II/B7-1/B7-2 e ativação de linfócitos T CD4+. As análises na região de inóculo mostraram o aumento no percentual de fibrócito na derme de camundongos após 15 dias de infecção tanto em animais C57BL/6 quanto em animais KO TLR-2. Neste sítio, os fibrócitos produziram citocinas e expressaram MHC-II, B7-1 e B7-2 podendo participar da resposta imune. As análises no linfonodo mostraram a existência de um alto percentual de fibrócitos nos animais controle, contudo, após infecção este percentual foi reduzido. Após infecção verificamos que os fibrócitos de animais WT C57BL/6 foram capazes de produzir as citocinas IL-4, IL-10 e IFN- durante o primeiro dia. Entretanto, na ausência de TLR-2 os fibrócitos presentes no linfonodo produziram TNF-α e IFN- que podem estar relacionadas com a ativação celular e aumento da capacidade de apresentação antigênica destas células durante a infecção. No linfonodo verificamos que os fibrócitos podem participar da apresentação antigênica após a infecção por L. (L.) amazonensis. Contudo, nos linfonodos de animais WT C57BL/6 observamos a redução significativa no percentual destas células expressando MHC-II e B7-1, o que pode estar relacionada a presença do TLR-2. Nos ensaios in vitro fibrócitos de camundongos C57BL/6 apresentaram alta capacidade endocítica, eliminaram os parasitas nas primeiras 24 horas de infecção, expressaram MHC-II/B7-1/B7-2 e foram capazes de ativar linfócitos T CD4+. Com isso, nossos resultados sugerem que os fibrócitos podem atuar na resposta celular e na apresentação antigênica durante a infecção por L. (L.) amazonensis, contudo estas funções podem ser moduladas pela participação do TLR-2 e pelo microambiente onde estes se encontram. / Fibrocytes were initially identified by their fast recruitment to the injured tissues and by acting directly on the immune response through recognition, antigen presentation and production of cytokines and chemokines. According to Grab et al., 2004 fibrocytes may participate in the immune response to leishmaniasis and therefore, in this study we propose to examine the cellular response and antigen presentation of fibrocytes in infection by L. (L.) amazonensis. In vivo experiments C57BL/6 and knockout receptor TLR-2 animals were inoculated in the ear dermis with 105 promastigotes of L. (L.) amazonensis and 1,7, 15 days after infection ears and lymph nodes were removed and processed for flow cytometry. For in vitro experiments peripheral blood fibrocytes from C57BL/6 mice were evaluated for their morphological and phenotypic, MHC-II/B7-1/B7-2 expression and ability to activate CD4+ T cells. The analyses in the inoculum region showed significant increase in the percentage of fibrocyte in the C57BL/6 and TLR-2 knockout mice after 15 days of infection. At this site fibrocytes part of the immune response by cytokines production and MHC-II, B7-1 and B7-2 expression. The analyses in the lymph node showed the existence of a high percentage of fibrocytes in the control animals, however, after infection, this percentage was reduced. After infection it was verified that the fibrocytes of C57BL/6 were able to produce cytokines IL-4, IL-10 and IFN- during the first days. However, in the absence of TLR-2 fibrocytes in the lymph produced TNF-α and IFN- that may be related to cell activation and increased antigen presentation capacity of these cells during infection. In the lymph node it was found that fibrocytes may participate in antigen presentation after infection with L. (L.) amazonensis. However, in lymph nodes of mice C57BL/6 it was observed the significant reduction in the percentage of those cells expressing MHC-II, B7-1, which can be related to the presence of the TLR-2. In vitro fibrocytes of C57BL/6 showed high endocytic capacity, eliminating the parasites within the first 24 hours after infection, and expressed MHC-II/B7-1/B7-2 being were able to activate CD4 + T cells. Thus, our results suggest that fibrocytes may act in cellular response and antigen presentation during infection by L. (L.) amazonensis, however these functions can be modulated by the involvement of TLR-2 and the in microenvironment.

Os fibrócitos foram inicialmente identificados pelo seu recrutamento rápido para os tecidos lesionados e por atuar diretamente na resposta imune através do reconhecimento, apresentação antigênica e produção de citocinas e quimiocinas. Segundo Grab (2004) fibrócitos podem participar da resposta imune na leishmaniaose e por isso no presente estudo propomos analisar a resposta celular e apresentação antigênica dos fibrócitos na infecção por L. (L.) amazonensis. Para os experimentos in vivo camundongos C57BL/6 e knockout para o receptor TLR-2 foram inoculados na derme auricular com 105 formas promastigotas de L. (L.) amazonensis e 1, 7, 15 dias após a infecção as regiões de inóculo e os linfonodos foram retirados e processados para citometria de fluxo. Para os experimentos in vitro fibrócitos foram obtidos de mononucleares do sangue periférico de camundongos C57BL/6. Os fibrócitos foram avaliados quanto às suas características morfológicas e fenotípicas, infecção, expressão de MHC-II/B7-1/B7-2 e ativação de linfócitos T CD4+. As análises na região de inóculo mostraram o aumento no percentual de fibrócito na derme de camundongos após 15 dias de infecção tanto em animais C57BL/6 quanto em animais KO TLR-2. Neste sítio, os fibrócitos produziram citocinas e expressaram MHC-II, B7-1 e B7-2 podendo participar da resposta imune. As análises no linfonodo mostraram a existência de um alto percentual de fibrócitos nos animais controle, contudo, após infecção este percentual foi reduzido. Após infecção verificamos que os fibrócitos de animais WT C57BL/6 foram capazes de produzir as citocinas IL-4, IL-10 e IFN- durante o primeiro dia. Entretanto, na ausência de TLR-2 os fibrócitos presentes no linfonodo produziram TNF-α e IFN- que podem estar relacionadas com a ativação celular e aumento da capacidade de apresentação antigênica destas células durante a infecção. No linfonodo verificamos que os fibrócitos podem participar da apresentação antigênica após a infecção por L. (L.) amazonensis. Contudo, nos linfonodos de animais WT C57BL/6 observamos a redução significativa no percentual destas células expressando MHC-II e B7-1, o que pode estar relacionada a presença do TLR-2. Nos ensaios in vitro fibrócitos de camundongos C57BL/6 apresentaram alta capacidade endocítica, eliminaram os parasitas nas primeiras 24 horas de infecção, expressaram MHC-II/B7-1/B7-2 e foram capazes de ativar linfócitos T CD4+. Com isso, nossos resultados sugerem que os fibrócitos podem atuar na resposta celular e na apresentação antigênica durante a infecção por L. (L.) amazonensis, contudo estas funções podem ser moduladas pela participação do TLR-2 e pelo microambiente onde estes se encontram. / Fibrocytes were initially identified by their fast recruitment to the injured tissues and by acting directly on the immune response through recognition, antigen presentation and production of cytokines and chemokines. According to Grab et al., 2004 fibrocytes may participate in the immune response to leishmaniasis and therefore, in this study we propose to examine the cellular response and antigen presentation of fibrocytes in infection by L. (L.) amazonensis. In vivo experiments C57BL/6 and knockout receptor TLR-2 animals were inoculated in the ear dermis with 105 promastigotes of L. (L.) amazonensis and 1,7, 15 days after infection ears and lymph nodes were removed and processed for flow cytometry. For in vitro experiments peripheral blood fibrocytes from C57BL/6 mice were evaluated for their morphological and phenotypic, MHC-II/B7-1/B7-2 expression and ability to activate CD4+ T cells. The analyses in the inoculum region showed significant increase in the percentage of fibrocyte in the C57BL/6 and TLR-2 knockout mice after 15 days of infection. At this site fibrocytes part of the immune response by cytokines production and MHC-II, B7-1 and B7-2 expression. The analyses in the lymph node showed the existence of a high percentage of fibrocytes in the control animals, however, after infection, this percentage was reduced. After infection it was verified that the fibrocytes of C57BL/6 were able to produce cytokines IL-4, IL-10 and IFN- during the first days. However, in the absence of TLR-2 fibrocytes in the lymph produced TNF-α and IFN- that may be related to cell activation and increased antigen presentation capacity of these cells during infection. In the lymph node it was found that fibrocytes may participate in antigen presentation after infection with L. (L.) amazonensis. However, in lymph nodes of mice C57BL/6 it was observed the significant reduction in the percentage of those cells expressing MHC-II, B7-1, which can be related to the presence of the TLR-2. In vitro fibrocytes of C57BL/6 showed high endocytic capacity, eliminating the parasites within the first 24 hours after infection, and expressed MHC-II/B7-1/B7-2 being were able to activate CD4 + T cells. Thus, our results suggest that fibrocytes may act in cellular response and antigen presentation during infection by L. (L.) amazonensis, however these functions can be modulated by the involvement of TLR-2 and the in microenvironment.

<p>The focus of this thesis was the role of fibrocytes in chronic lung disease. These bone marrow derived cells have been identified in the lung and the circulation in patient samples and animal models of lung injury. However, the precise mechanistic role of the fibrocyte is still to be elucidated.</p> <p>Live assessment of lung changes in animal models of chronic lung disease allows for real time observation of changes, and gives a readout which can be translated to humans who undergo similar tests. In this thesis, we adapted an existing model of lung injury, and delivered a discrete treatment to a single lung lobe while monitoring its successful delivery.</p> <p>I also developed a robust system to examine the relationship between fibrocyte response, and cytokine expression previously identified in chronic lung disease. We found a connection between cytokine expression and fibrocyte mobilisation. Our model showed that fibrocyte mobilisation in the presence of existing lung injury does not improve and rather can worsen existing lung injury. This was a significant finding as it confirms the role of the fibrocyte as a participator or conductor in fibrogenesis and it suggests that this cell may play a role in the development of chronic lung diseases.</p> <p>Finally, we contributed to the ongoing characterisation of the fibrocyte as a prospective biomarker. We confirmed the cell’s identity by characterising it by its known markers and biological characteristics. We also identified the presence of this cell in chronic lung disease and linked its presence to disease progression.</p> / Doctor of Philosophy (Medical Science)

Fibrotic diseases have a poor prognosis with no FDA approved therapies. Monocyte-derived, fibroblast-like cells called fibrocytes participate in the formation of fibrotic lesions. The conserved pentraxin protein serum amyloid P (SAP) inhibits fibrocyte differentiation in cell culture, and injections of SAP significantly reduce fibrosis in several animal models. SAP binds to the receptors for the Fc portion of immunoglobulin G (FcγR), and has been crystallized bound to FcγRIIa. The in vivo activity of SAP appears to be dependent on the common γ chain (FcγR) of activating Fc receptors. The goal of my project is to elucidate the functional domains of SAP and the receptor responsible for SAP bioactivity, which could lead to refinements for SAP as a therapeutic agent and additional drug targets. I found that mutagenesis of the residues critical for SAP binding to FcγRIIa only moderately decreases SAP's ability to inhibit fibrocyte differentiation. In murine cells, deletion of FcγR or FcγRI significantly reduced sensitivity to SAP. Deletion of the combination of FcγRIIb/FcγRIIIa/FcγRIV did not significantly affect sensitivity to SAP, while deletion of just the inhibitory receptor FcγRIIb increased sensitivity to SAP. In human cells, siRNA-mediated reduction of FcγR or FcγRI levels significantly decreased sensitivity to SAP, while reduction of FcγRIIb levels increased sensitivity to SAP. These observations suggest that SAP, at least in part, uses FcγRI and FcγR to inhibit fibrocyte differentiation. I am also interested in how SAP functions in various disease states. SAP is known to be elevated in Alzheimer's disease (AD) and binds to amyloid plaques in the brain, a key hallmark of AD. There is a significant population of individuals that have key hallmarks of AD but show no signs of cognitive impairment, termed non-demented with AD neuropathology (NDAN). I evaluated SAP levels in post mortem samples of hippocampus and frontal cortex in age-matched controls, AD, and NDAN individuals. AD individuals had significantly increased SAP levels, while NDAN samples had no significant difference in SAP levels compared to controls. These results suggest that low levels of SAP in plaques marks the brains of individuals that escape dementia despite the presence of beta amyloid plaques.

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.