@phdthesis{Andelovic2024, author = {Andelovic, Kristina}, title = {Characterization of arterial hemodynamics using mouse models of atherosclerosis and tissue-engineered artery models}, doi = {10.25972/OPUS-30360}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-303601}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {Within this thesis, three main approaches for the assessment and investigation of altered hemodynamics like wall shear stress, oscillatory shear index and the arterial pulse wave velocity in atherosclerosis development and progression were conducted: 1. The establishment of a fast method for the simultaneous assessment of 3D WSS and PWV in the complete murine aortic arch via high-resolution 4D-flow MRI 2. The utilization of serial in vivo measurements in atherosclerotic mouse models using high-resolution 4D-flow MRI, which were divided into studies describing altered hemodynamics in late and early atherosclerosis 3. The development of tissue-engineered artery models for the controllable application and variation of hemodynamic and biologic parameters, divided in native artery models and biofabricated artery models, aiming for the investigation of the relationship between atherogenesis and hemodynamics Chapter 2 describes the establishment of a method for the simultaneous measurement of 3D WSS and PWV in the murine aortic arch at, using ultra high-field MRI at 17.6T [16], based on the previously published method for fast, self-navigated wall shear stress measurements in the murine aortic arch using radial 4D-phase contrast MRI at 17.6 T [4]. This work is based on the collective work of Dr. Patrick Winter, who developed the method and the author of this thesis, Kristina Andelovic, who performed the experiments and statistical analyses. As the method described in this chapter is basis for the following in vivo studies and undividable into the sub-parts of the contributors without losing important information, this chapter was not split into the single parts to provide fundamental information about the measurement and analysis methods and therefore better understandability for the following studies. The main challenge in this chapter was to overcome the issue of the need for a high spatial resolution to determine the velocity gradients at the vascular wall for the WSS quantification and a high temporal resolution for the assessment of the PWV without prolonging the acquisition time due to the need for two separate measurements. Moreover, for a full coverage of the hemodynamics in the murine aortic arch, a 3D measurement is needed, which was achieved by utilization of retrospective navigation and radial trajectories, enabling a highly flexible reconstruction framework to either reconstruct images at lower spatial resolution and higher frame rates for the acquisition of the PWV or higher spatial resolution and lower frame rates for the acquisition of the 3D WSS in a reasonable measurement time of only 35 minutes. This enabled the in vivo assessment of all relevant hemodynamic parameters related to atherosclerosis development and progression in one experimental session. This method was validated in healthy wild type and atherosclerotic Apoe-/- mice, indicating no differences in robustness between pathological and healthy mice. The heterogeneous distribution of plaque development and arterial stiffening in atherosclerosis [10, 12], however, points out the importance of local PWV measurements. Therefore, future studies should focus on the 3D acquisition of the local PWV in the murine aortic arch based on the presented method, in order to enable spatially resolved correlations of local arterial stiffness with other hemodynamic parameters and plaque composition. In Chapter 3, the previously established methods were used for the investigation of changing aortic hemodynamics during ageing and atherosclerosis in healthy wild type and atherosclerotic Apoe-/- mice using the previously established methods [4, 16] based on high-resolution 4D-flow MRI. In this work, serial measurements of healthy and atherosclerotic mice were conducted to track all changes in hemodynamics in the complete aortic arch over time. Moreover, spatially resolved 2D projection maps of WSS and OSI of the complete aortic arch were generated. This important feature allowed for the pixel-wise statistical analysis of inter- and intragroup hemodynamic changes over time and most importantly - at a glance. The study revealed converse differences of local hemodynamic profiles in healthy WT and atherosclerotic Apoe-/- mice, with decreasing longWSS and increasing OSI, while showing constant PWV in healthy mice and increasing longWSS and decreasing OSI, while showing increased PWV in diseased mice. Moreover, spatially resolved correlations between WSS, PWV, plaque and vessel wall characteristics were enabled, giving detailed insights into coherences between hemodynamics and plaque composition. Here, the circWSS was identified as a potential marker of plaque size and composition in advanced atherosclerosis. Moreover, correlations with PWV values identified the maximum radStrain could serve as a potential marker for vascular elasticity. This study demonstrated the feasibility and utility of high-resolution 4D flow MRI to spatially resolve, visualize and analyze statistical differences in all relevant hemodynamic parameters over time and between healthy and diseased mice, which could significantly improve our understanding of plaque progression towards vulnerability. In future studies the relation of vascular elasticity and radial strain should be further investigated and validated with local PWV measurements and CFD. Moreover, the 2D histological datasets were not reflecting the 3D properties and regional characteristics of the atherosclerotic plaques. Therefore, future studies will include 3D plaque volume and composition analysis like morphological measurements with MRI or light-sheet microscopy to further improve the analysis of the relationship between hemodynamics and atherosclerosis. Chapter 4 aimed at the description and investigation of hemodynamics in early stages of atherosclerosis. Moreover, this study included measurements of hemodynamics at baseline levels in healthy WT and atherosclerotic mouse models. Due to the lack of hemodynamic-related studies in Ldlr-/- mice, which are the most used mouse models in atherosclerosis research together with the Apoe-/- mouse model, this model was included in this study to describe changing hemodynamics in the aortic arch at baseline levels and during early atherosclerosis development and progression for the first time. In this study, distinct differences in aortic geometries of these mouse models at baseline levels were described for the first time, which result in significantly different flow- and WSS profiles in the Ldlr-/- mouse model. Further basal characterization of different parameters revealed only characteristic differences in lipid profiles, proving that the geometry is highly influencing the local WSS in these models. Most interestingly, calculation of the atherogenic index of plasma revealed a significantly higher risk in Ldlr-/- mice with ongoing atherosclerosis development, but significantly greater plaque areas in the aortic arch of Apoe-/- mice. Due to the given basal WSS and OSI profile in these two mouse models - two parameters highly influencing plaque development and progression - there is evidence that the regional plaque development differs between these mouse models during very early atherogenesis. Therefore, future studies should focus on the spatiotemporal evaluation of plaque development and composition in the three defined aortic regions using morphological measurements with MRI or 3D histological analyses like LSFM. Moreover, this study offers an excellent basis for future studies incorporating CFD simulations, analyzing the different measured parameter combinations (e.g., aortic geometry of the Ldlr-/- mouse with the lipid profile of the Apoe-/- mouse), simulating the resulting plaque development and composition. This could help to understand the complex interplay between altered hemodynamics, serum lipids and atherosclerosis and significantly improve our basic understanding of key factors initiating atherosclerosis development. Chapter 5 describes the establishment of a tissue-engineered artery model, which is based on native, decellularized porcine carotid artery scaffolds, cultured in a MRI-suitable bioreactor-system [23] for the investigation of hemodynamic-related atherosclerosis development in a controllable manner, using the previously established methods for WSS and PWV assessment [4, 16]. This in vitro artery model aimed for the reduction of animal experiments, while simultaneously offering a simplified, but completely controllable physical and biological environment. For this, a very fast and gentle decellularization protocol was established in a first step, which resulted in porcine carotid artery scaffolds showing complete acellularity while maintaining the extracellular matrix composition, overall ultrastructure and mechanical strength of native arteries. Moreover, a good cellular adhesion and proliferation was achieved, which was evaluated with isolated human blood outgrowth endothelial cells. Most importantly, an MRI-suitable artery chamber was designed for the simultaneous cultivation and assessment of high-resolution 4D hemodynamics in the described artery models. Using high-resolution 4D-flow MRI, the bioreactor system was proven to be suitable to quantify the volume flow, the two components of the WSS and the radStrain as well as the PWV in artery models, with obtained values being comparable to values found in literature for in vivo measurements. Moreover, the identification of first atherosclerotic processes like intimal thickening is achievable by three-dimensional assessment of the vessel wall morphology in the in vitro models. However, one limitation is the lack of a medial smooth muscle cell layer due to the dense ECM. Here, the utilization of the laser-cutting technology for the generation of holes and / or pits on a microscale, eventually enabling seeding of the media with SMCs showed promising results in a first try and should be further investigated in future studies. Therefore, the proposed artery model possesses all relevant components for the extension to an atherosclerosis model which may pave the way towards a significant improvement of our understanding of the key mechanisms in atherogenesis. Chapter 6 describes the development of an easy-to-prepare, low cost and fully customizable artery model based on biomaterials. Here, thermoresponsive sacrificial scaffolds, processed with the technique of MEW were used for the creation of variable, biomimetic shapes to mimic the geometric properties of the aortic arch, consisting of both, bifurcations and curvatures. After embedding the sacrificial scaffold into a gelatin-hydrogel containing SMCs, it was crosslinked with bacterial transglutaminase before dissolution and flushing of the sacrificial scaffold. The hereby generated channel was subsequently seeded with ECs, resulting in an easy-to-prepare, fast and low-cost artery model. In contrast to the native artery model, this model is therefore more variable in size and shape and offers the possibility to include smooth muscle cells from the beginning. Moreover, a custom-built and highly adaptable perfusion chamber was designed specifically for the scaffold structure, which enabled a one-step creation and simultaneously offering the possibility for dynamic cultivation of the artery models, making it an excellent basis for the development of in vitro disease test systems for e.g., flow-related atherosclerosis research. Due to time constraints, the extension to an atherosclerosis model could not be achieved within the scope of this thesis. Therefore, future studies will focus on the development and validation of an in vitro atherosclerosis model based on the proposed bi- and three-layered artery models. In conclusion, this thesis paved the way for a fast acquisition and detailed analyses of changing hemodynamics during atherosclerosis development and progression, including spatially resolved analyses of all relevant hemodynamic parameters over time and in between different groups. Moreover, to reduce animal experiments, while gaining control over various parameters influencing atherosclerosis development, promising artery models were established, which have the potential to serve as a new platform for basic atherosclerosis research.}, subject = {H{\"a}modynamik}, language = {en} } @phdthesis{Knochenhauer2023, author = {Knochenhauer, Tim}, title = {Die Rolle von HIF-1α in T-Zellen bei kardiovaskul{\"a}ren Erkrankungen}, doi = {10.25972/OPUS-32275}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-322758}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Die Atherosklerose ist als Ursache kardiovaskul{\"a}rer Erkrankungen, welche die h{\"a}ufigste Todesursache weltweit darstellen, von großer klinischer und wissenschaftlicher Relevanz. Atherosklerose ist charakterisiert durch Einlagerungen von Lipiden in die Gef{\"a}ßwand, welche zur Ausbildung von Plaques f{\"u}hren. Als Folge wird eine chronische Entz{\"u}ndungsreaktion eingeleitet, die durch spezifische Immunzellen, unter anderem T-Lymphozyten, und komplexe molekulare Prozesse aufrechterhalten wird. Durch eine verminderte Sauerstoffdiffusionskapazit{\"a}t und eine hohe Zelldichte ist das Milieu in den Plaques hypoxisch. Zur zellul{\"a}ren Anpassung an ein solches hypoxisches Milieu werden Hypoxie-induzierbare Faktoren (HIF) in den Immunzellen stabilisiert. Der Transkriptionsfaktor HIF-1 ist ein heterodimeres Protein, welches die Transkription bestimmter Zielgene initiiert, die den Zellen notwendige Adaptationen des Zellstoffwechsels an ein vermindertes Sauerstoffangebot erm{\"o}glichen. Das Ziel der vorliegenden Arbeit bestand darin zu untersuchen, inwiefern sich ein Ausschalten des Transkriptionsfaktor HIF-1α selektiv in T-Lymphozyten auf Atherosklerose und Myokardinfarkt auswirkt. Die funktionelle Bedeutung von HIF-1α in T-Zellen in der Pathogenese dieser Erkrankungen wurde an zwei Mausmodellen untersucht. Im Atherosklerose Modell wurde Biomaterial von LDLR-/- M{\"a}usen mit T-Zell spezifischem Knockout von HIF-1α nach achtw{\"o}chiger fettreicher Western-Typ Di{\"a}t untersucht. Histologisch zeigte sich eine vermehrte Plaqueauspr{\"a}gung und ein verminderter Makrophagenanteil in den Plaques. Durchflusszytometrisch und mittels qPCR konnten keine Unterschiede in der Lymphozytendifferenzierung in Milz und Lymphknoten dieser M{\"a}use nachgewiesen werden. Im Myokardinfarkt-Modell mit T-Zell spezifischem HIF-1α Knockout konnte in fr{\"u}heren Untersuchungen der Arbeitsgruppe eine vergr{\"o}ßerte Infarktzone mit eingeschr{\"a}nkter kardialer Funktion nachgewiesen werden. Histologisch konnte im Rahmen dieser Arbeit hierf{\"u}r kein zellmorphologisches Korrelat in Kardiomyozytengr{\"o}ße oder der Vaskularisation des Myokards gefunden werden. In Zukunft k{\"o}nnte HIF-1α in T-Lymphozyten ein m{\"o}glicher Angriffspunkt zur medikament{\"o}sen Pr{\"a}vention oder Therapie kardiovaskul{\"a}rer Erkrankungen sein.}, subject = {Hypoxie-induzierbarer Faktor}, language = {de} } @phdthesis{Schneider2023, author = {Schneider, Nicole}, title = {Untersuchung der Expression von SET7 und anderer epigenetischer Enzyme in vitro und vivo im Modell der Atherosklerose}, doi = {10.25972/OPUS-32895}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-328952}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Bei der Atherosklerose handelt es sich um eine chronische inflammatorische Erkrankung, die sich an der arteriellen Gef{\"a}ßinnenwand abspielt. Ihre Haupt-Manifestationsformen Schlaganfall und Herzinfarkt z{\"a}hlen zu den h{\"a}ufigsten Todesursachen weltweit. Eine chronische Endothelbelastung und -funktionsst{\"o}rung, beeinflusst durch Risikofaktoren wie Diabetes, arterieller Bluthochdruck, Rauchen und Entz{\"u}ndungszust{\"a}nde, f{\"u}hren zur Permeabilit{\"a}tserh{\"o}hung des Endothels, zur Zelleinwanderung, subendothelialen Lipidanreicherung, Migration glatter Muskelzellen und der Ausbildung atherosklerotischer L{\"a}sionen. Es kommt zu Aktivierung des Immunsystems und fortschreitender Entz{\"u}ndungsreaktion, schließlich zur Ausbildung eines nekrotischen Kerns und zunehmender Vulnerabilit{\"a}t des Plaques. Epigenetische Ver{\"a}nderungen betreffen klassischerweise das Chromatinger{\"u}st. Durch DNA-Methylierung und -Demethylierung sowie verschiedene Modifikationen der Histon-Proteine kann die DNA in ihrer Zug{\"a}nglichkeit ver{\"a}ndert werden. So kann die Transkription eines bestimmten Genes direkt und potenziell l{\"a}ngerfristig beeinflusst werden, ohne dass Alterationen der DNA-Basenfolge selbst stattfinden. Das Enzym SET7 nimmt hierbei eine Sonderrolle ein, da es neben einer Methylierung von Histon 3 auch verschiedene zellul{\"a}re Zielstrukturen posttranslational direkt methylieren kann. Epigenetische Ver{\"a}nderungen im Kontext der Atherosklerose sind bereits vereinzelt beschrieben. Auch sind sie relevant in der Reaktion auf Umwelteinfl{\"u}sse und bei inflammatorischen Vorg{\"a}ngen. Der Frage, ob epigenetische Mechanismen im atherosklerotischen Geschehen eine Rolle spielen, sollte in dieser Arbeit nachgegangen werden. Dazu wurde in Zellkulturversuchen f{\"u}r Makrophagen und glatte Muskelzellen gepr{\"u}ft, ob die einzelnen pro-atherosklerotischen Stimuli oxLDL, IL-1β, TNFα und LPS bereits zu relevanten Ver{\"a}nderungen epigenetischer Enzyme f{\"u}hren. Dies erfolgte {\"u}ber Vergleich der entsprechenden mRNA mittels qPCR. Zur Untersuchung der genaueren Dynamik wurde f{\"u}r die Enzyme SET7 und DNMT1 der zeitliche Ablauf dieser Reaktion auf TNFα-Stimulation in Makrophagen genauer betrachtet. Unter gleichen Versuchsbedingungen wurde außerdem die {\"A}nderung der mRNA-Expression einiger Matrixmetalloproteasen, TIMP-Enzyme, Zytokine und Transkriptionsfaktoren analysiert,um zuk{\"u}nftig kausale Zusammenh{\"a}nge weiter aufdecken zu k{\"o}nnen. Auch die Frage nach Ver{\"a}nderungen epigenetischer Enzyme in der Ldlr-/--Maus nach fettreicher Di{\"a}t im Vergleich zu Ldlr-/--M{\"a}usen ohne Di{\"a}t sollte hier beantwortet werden. Dazu wurde die mRNA der Zellsuspensionen aus Milz, Aortenwurzel und gesamter Aorta der Tiere mithilfe der qPCR verglichen. Schließlich sollte ein effizienter Weg f{\"u}r einen individuellen und flexiblen SET7 knock-out etabliert werden, um weitere Studien dieses Enzyms zu erm{\"o}glichen. Hierzu wurde die Methode des CRISPR/Cas9 Systems gew{\"a}hlt und abschließend die Funktionalit{\"a}t des Systems {\"u}berpr{\"u}ft.}, subject = {Arteriosklerose}, language = {de} }