Vulnerable plaque is a clinically silent condition of atherosclerotic plaque that leaves a large number of patients at risk of a coronary event. A method to detect vulnerable atherosclerotic plaque would greatly enhance the ability of clinicians to diagnose and treat patients at risk. Fluorescence lifetime imaging microscopy (FLIM) offers a way to extract both spatial and biochemical information from plaque by taking several wide-field images over time. The goal of this study was to determine the potential of a FLIM angioscopy system to detect and differentiate coronary atherosclerotic plaques ex-vivo into several groups including thin, fibrotic, lipid-laden, thick-cap fibroatheroma (FA), and fibrocalcified. Samples were extracted post-mortem weekly and sliced open to have their lumens imaged. For each sample, 51 time resolved wide-field images were taken over 10 nanoseconds at 390 (±40) nm, 450 (±40) nm, and 550 (±88) nm wavelengths. To analyze the samples, the intensity map and lifetime map were created at each wavelength. The intensity map was simply the wide-field images summed in time and normalized. In order to calculate lifetime at each point, a fast, model-free Laguerre deconvolution algorithm was recently developed for FLIM data analysis and was used. This allowed for fast, efficient estimations of the fluorescence decay curves at each pixel of the FLIM images and facilitated the computation of quantitative parameters describing the fluorescence emission of the tissue, specifically, the relative fluorescence intensity and lifetime at defined emission bands. Statistical analysis on these FLIM derived parameters indicated that the autofluorescence emission of the plaques allows for distinguishing relative plaque thickness: thin plaque, whose signal is dominated by elastin fluorophores, shows a marked difference between thicker plaques, such as fibrotic, fibrocalcified and thick-cap FA (who are dominated primarily by collagen). However, the ability of the current FLIM system to differentiate vulnerable plaque remains in question due to the absence of thin-cap FA samples. Further work has also been proposed; of primary concern is gathering thin-cap FA plaque samples needed to validate the system’s ability to differentiate vulnerable plaques from other common groupings.

Atherosclerotic plaque ruptures have been determined as the most common underlying cause of acute coronary syndromes and stroke. Currently, the standard of care for plaque rupture risk is based on the amount of luminal stenosis presented in a particular vessel; however, X-ray angiographic studies have shown that plaques at risk of rupture generally show <50% luminal narrowing. These findings explicate the need for other, more accurate methods of identifying problem lesions prior to the rupture event. Unfortunately, the study of thrombotic events and vulnerable plaque lesions in humans is difficult due to the spontaneity of rupture and the lengthy time course of disease progression. To further the understanding of plaque rupture risk in light of vulnerability detection, a rabbit model of atherothrombosis was used in conjunction with magnetic resonance imaging (MRI). MRI has been validated as a suitable imaging modality for in vivo, non-invasive detection of atherosclerosis and has provided quantitative predictors of plaques at risk of rupture. Additionally, the rabbit model has been shown, histologically, to present 6 of the 8 human plaque types classified by the American Heart Association. The first portion of this dissertation work focuses on using MRI to serially image rabbits undergoing the atherosclerotic protocol in order to assess rupture risk at the various time points. Previous work has determined that an increase in the vessel remodeling ratio (which hides a large plaque in the vessel wall) and contrast uptake (which indicates inflammation) are both characteristics of increased rupture risk. By obtaining these parameters at various time points in the disease progression, it was possible to determine when a certain plaque displays a heightened risk of rupture. The second portion of this work tested the efficacy of a pro-resolving molecule, lipoxin (an endogenous molecule), in reducing atherosclerotic disease state, specifically rupture with a luminal thrombus. Using chronic administration of this molecule in the same rabbit model of atherosclerosis yielded a faint reduction in atherosclerotic severity based on the parameters of decreased vessel lipid content and decreased thrombotic events presented in the treated group.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20621号 / 医博第4270号 / 新制||医||1023(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 木村 剛, 教授 渡邉 大 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Phase-change contrast agents (PCCAs) are an innovative form of imaging agent with practical applications in both the research and clinical settings. PCCAs are derived from gaseous microbubbles, which are able to act as targeted-contrast agents through conjugation of a ligand that is selective for an overexpressed receptor or biomarker in a given disease. Gaseous microbubbles can be condensed to liquid phase nanodroplets, which should be sufficiently small to extravasate into cells and/or tissues given their size and stability. Once liquid nanodroplets have internalized within a given tissue, they can be "activated" back into gaseous microbubbles with ultrasound at clinically used frequencies and energy outputs. This is purposeful as microbubbles provide much greater ultrasound reflectivity than nanodroplets. In this study, PCCAs and/or microbubbles act as a targeting agent in multiple scenarios. The projects in this study include- examination of binding and internalization of targeted PCCAs with different gaseous cores within MDA-MB-231 breast cancer cells, vaporization of liquid phase nanodroplets through application of acoustic energy via focused ultrasound (FUS), and targeting vulnerable plaque in the heart with different types of targeted microbubbles under varying shear-stresses.

INTRODUCTION: The“vulnerable plaque" is defined as the“precursor lesion" that ultimately ends in acute coronary thrombi (clots) that create a heart attack. Macrophages and inflammatory cells, found preferentially in vulnerable plaque, sustain their activity in the plaque through anaerobic metabolism and lactate production. The ultimate goal is to assess anaerobic metabolism in-vivo by measuring tissue pH and lactate concentration in atherosclerotic plaques using optical spectroscopy. The proposed in-vitro optical probe design, experimental method, and spectroscopic data analysis methodology are established in this research. METHODS: A fiber optic probe was designed and built based on both Monte Carlo simulations and bench testing with the goal to collect light from a small volume of tissue. A simulation of the depth penetration of the proposed probe was performed on normal and atherosclerotic aortic tissue, and the final probe was bench tested using normal aorta. A method was developed to preserve plaque metabolic status of tissue harvested from patients. Human atherosclerotic tissue obtained immediately after carotid endarterectomy was placed in Minimum Essential Medium (MEM) with non-essential amino acids supplement, bubbled with 75%O2/20%N2/5%CO2 at 37°C. Tissue pH, pCO2, pO2 and temperature with (n=7) and without (n=2) the media preparation over time were reviewed to assess plaque viability and maintenance of physiological conditions. Additional plaques placed in media were used for development of chemometric methods to measure pH and lactate. Areas of each plaque were randomly chosen for analysis. Reflectance spectra were collected with a dispersive spectrometer (400-1100 nm) and a Fourier-transform near-infrared spectrometer (1100-2400 nm) using the fiber optic probe. Reference measurements for tissue pH and lactate were made with glass microelectrodes and micro-enzymatic assay, respectively. Partial least-squares (PLS) data analysis was used to develop multivariate calibration models on an initial set of 5-6 plaques relating the optical spectra to the reference tissue pH (n=20) or the lactate concentration (n=21) to assess data quality. The coefficient of multiple determination (R2), the standard error of cross-validation (SECV), and the number of factors were used to assess the model performance. Additional points were collected from ~14 plaques and added to preliminary data. Pre-processing techniques were then used to see if preliminary data results could be improved by reducing different sources of variability with the introduction of more points. RESULTS: Monte Carlo simulations and depth penetration tests with the final probe design showed light is collected from ~1 mm3 volume of tissue using a 50 micron source-receiver separation. Tissue pH, pCO2, pO2 and temperature values demonstrated that the plaques were viable and stable in the media preparation for a maximum of 4 hours. Data from the first six plaques collected for lactate analysis showed that for seventeen points, a six-factor model produced adequate results (R2=0.83 SECV=1.4 micromoles lactate/gram tissue). Data from the first five plaques collected for tissue pH analysis, showed for seventeen different points, a three-factor model produced adequate results (R2=0.75 SECV=0.09 pH units). When additional points were added to either data set, model results were degraded. CONCLUSIONS: The in-vitro optical probe design and experimental procedures was established and the feasibility of the optical method demonstrated with preliminary data. However, with the addition of more data points, different sources of tissue and spectral variability were observed to affect calibration. The gross pathology type and mismatched optical volume to reference measurement volume limited the tissue pH determination. The reference measurement precision, the spatial resolution of the reference lactate measurement, and unmodeled tissue variability (water and proteins) limited the lactate determination. Large variability in all optical measurements was observed. Additional in-vitro data collection would be required such that the variability due to the tissue is reduced and any spectrometer variability adequately compensated to be able to use the optical calibration in-vivo.

La maladie coronaire représente un problème majeur de santé publique. Sa manifestation la plus sévère, le syndrome coronarien aigu, est en grande majorité lié à la rupture de plaques d'athérome vulnérables. Les travaux effectués dans le cadre de cette thèse portent sur l'étude pré-clinique de la plaque d'athérome dans un modèle murin : la souris déficiente en apolipoprotéine E (apoE-/-).La rupture des plaques d'athérome vulnérables survient à la suite d'une élévation des contrainte mécaniques au niveau de la capsule fibreuse. Une première partie de ce travail de thèse porte sur l'évaluation de la contrainte présente dans la paroi des vaisseaux de souris apoE-/- porteuses de lésions athéromateuses. Ces lésions présentent une faible rigidité et en conséquence de faibles contraintes. Ces observations donnent un élément d'explication de l'absence de rupture dans ce modèle animal.Les plaques d'athérome vulnérables présentent une inflammation importante ; l'imagerie de l'inflammation vasculaire est une approche pertinente pour l'identification des plaques d'athérome vulnérables. Une seconde partie de ce travail de thèse porte sur l'imagerie de l'athérosclérose en ciblant le processus inflammatoire chez la souris apoE-/- avec d'une part l'évaluation de radiotraceurs peptidiques développés au sein du laboratoire et décrits comme ligands de la molécule d'adhésion VCAM-1, surexprimée dans le contexte inflammatoire, et d'autre part l'évaluation du [18F]-fluorodésoxyglucose (FDG), décrit pour s'accumuler dans les cellules inflammatoires et le tissu adipeux brun. L'évaluation poussée des peptides originaux a mis en évidence un manque de robustesse dans leur comportement biologique. D'autre part, une captation similaire de FDG a été observée dans les lésions athéromateuses et du tissu le brun péri-aortique se trouvant à proximité chez la souris apoE-/- . Cette limitation souligne la nécessité de l'optimisation des procédés expérimentaux destinés à l'évaluation de l'inflammation des lésions d'athérosclérose avec le FDG dans ce modèle animal. / Coronary artery disease is a major healthcare issue. Acute coronary syndrome is mainly caused by the rupture of vulnerable coronary plaques. This thesis focused on the preclinical study of atherosclerosis in a mouse model: the apolipoprotein E-deficient mouse (apoE-/-).Vulnerable plaque rupture is caused by an elevation of mechanical stress in the fibrous cap of atherosclerotic plaques. The first part of this thesis focused on the parietal stress evaluation in atheromatous lesions from apoE-/- mouse vessels. Atherosclerotic tissue showed low stiffness resulting in low levels of mechanical stress in atheromatous lesions. The low level of mechanical stress might account for the atherosclerotic plaque stability in this animal model.Rupture-prone vulnerable atheromatous plaques are highly inflammatory; vascular inflammation imaging appears therefore as a relevant strategy for vulnerable plaque imaging. The second part of this thesis dealt with atherosclerosis imaging in the apoE-/- mouse model, firstly with the evaluation of original peptidic radiotracers described as a VCAM-1 binders, an adhesion molecule over-expressed during inflammation, and secondly with the evaluation of [18F]-fluorodeoxyglucose (FDG), which was shown to accumulate in inflammatory cells and in the brown adipose tissue (BAT). Advanced evaluation of peptides revealed a lack of robustness in their biological behavior. A similar FDG uptake was observed in the atherosclerotic lesions and in the periaortic brown adipose tissue of apoE-/- mouse found in the vicinity. This potential confounding factor emphasizes the need to carefully design preclinical studies using FDG for the evaluation of lesion inflammation in this animal model.

La thrombose est souvent liée à la survenue d’une rupture de la plaque d’athérosclérose qui expose le tissu sous endothélial thrombogène aux plaquettes sanguines circulantes. L’un des problèmes actuels est l’identification des mécanismes qui sont à l’origine de la fracture. Nous avons testé l’hypothèse que la vasoconstriction, induite par le LTC4 ou le thromboxane A2 (TXA2), joue un rôle dans la rupture de plaques d’athérosclérose et dans la formation de l’athérothrombose. Dans ce travail nous avons adapté la technique classique de la microscopie électronique pour observer les ruptures survenues sur une plaque d’athérosclérose. Avec cette méthode, nous avons montré qu’une vasoconstriction peut engendrer des ruptures de plaques, mais seulement lorsque ces plaques sont fragilisées (dites ‘vulnérables’) soit par un régime riche en graisse ou après exposition de la souris à un lipopolysaccharide extrait de membranes bactériennes. Nous avons montré aussi que la plaque produit du TXA2, et que l’absence de son récepteur chez les souris apoE-/- déficientes en TP prévient la rupture des plaques vulnérabilisées. La vasoconstriction constitue donc un élément déterminant dans la rupture des plaques vulnérables. Les résultats de ce travail identifient le TXA2 comme étant une éventuelle cible thérapeutique pour prévenir les ruptures des plaques. D’autre part, les analyses par microscopie électronique à balayage de la thrombose in vivo, et les analyses en microscopie électronique en transmission (MET) de l’ultrastructure des plaquettes agrégées in vitro, ont montré que les plaquettes activées fusionnent entre elle permettant ainsi la cicatrisation de l’endothélium lésé. / Rupture of atherosclerotic plaques exposes sub-endothelial thrombogenic material to circulating platelets, frequently leads to thrombus formation. At present, the mechanisms behind plaque ruptures are not well understood. We studied the hypothesis that vasoconstriction induced by LTC4 or thromboxane A2 (TXA2) plays an important role in plaque rupture and formation of atherothombosis. We used scanning electron microscopy to study rupture of the plaque in detail. We were able to show that vasoconstriction leads to plaque rupture, but only after plaques have been vulnerabilized by high fat diet, or by exposure of mice to lipopolysaccharide extracted from bacterial membranes. We could also show that plaques produce TXA2. As consequence the absence of TP, a receptor for TXA2 in apoE-/- mice prevented rupture of vulnerable plaques. Vasoconstriction is hence a determining factor in the rupture of vulnerable plaques. Our work has identified TXA2 as a potential therapeutic target in preventing plaque rupture. Moreover, analysis of the thrombosis in vivo by scanning electron microscopy, and the in vitro study of the ultra structure of aggregated platelets by transmission electron microscopy, showed that activated platelets fuse together to heal the injured endothelium.

No description available.

Il est maintenant admis que la composition de la plaque athérosclérotique est un déterminant majeur de sa vulnérabilité à se rompre. Vu que la composition de la plaque affecte ses propriétés mécaniques, l'évaluation locale des propriétés mécaniques de la plaque d'athérome peut nous informer sur sa vulnérabilité. L'objectif est de comparer les techniques d’élastographie ultrasonores endovasculaire (EVE) et non-invasive (NIVE) en fonction de leur potentiel à identifier les composantes calcifiées et lipidiques de la plaque. Les acquisitions intravasculaire et extravasculaire ont été effectuées sur les artères carotidiennes de neuf porcs hypercholestérolémiques à l’aide d’un cathéter de 20 MHz et d'une sonde linéaire de 7.5 MHz, respectivement. Les valeurs de déformation radiale et axiale, rapportés par EVE et NIVE, ont été corrélées avec le pourcentage des zones histologiques calcifiées et lipidiques pour cinq plaques. Nos résultats démontrent une bonne corrélation positive entre les déformations et les composantes calcifiées (r2 = 0.82, P = 0.034 valeur par EVE et r2 = 0.80, P = 0.041 valeur par NIVE). Une forte corrélation entre les déformations axiales et les contenus lipidiques par NIVE (r2 = 0.92, P-value = 0.010) a été obtenue. En conclusion, NIVE et EVE sont des techniques potentielles pour identifier les composants de la plaque et aider les médecins à diagnostiquer précocement les plaques vulnérables. / It is now widely accepted that plaque composition is a major determinant of plaque’s vulnerability to rupture. Since composition of the plaque affects its mechanical properties, the local assessment of mechanical properties of atherosclerotic plaque may inform us about plaque’s vulnerability. The objective is to compare ultrasonic endovascular elastography (EVE) versus non-invasive vascular elastography (NIVE) according to their potential to identify plaque contents. Intravascular and extravascular acquisitions were performed on carotid arteries of nine hypercholesterolemic minipigs with a 20 MHz catheter and a 7.5 MHz standard probe, respectively. Radial and axial strain values, reported by EVE and NIVE respectively, were correlated with histological area of lipid and calcium for five plaques. Our results demonstrate a good positive correlation between strains and calcified contents (r2=0.82, P-value=0.034 by EVE and r2=0.80, P-value= 0.041 by NIVE). Additionally, there is a strong correlation between axial strains and lipid contents by NIVE (r2=0.92, P-value= 0.010). In conclusion, NIVE and EVE are the potential techniques to identify plaque components and to help physicians to early diagnose the vulnerable plaques.

Il est maintenant admis que la composition de la plaque athérosclérotique est un déterminant majeur de sa vulnérabilité à se rompre. Vu que la composition de la plaque affecte ses propriétés mécaniques, l'évaluation locale des propriétés mécaniques de la plaque d'athérome peut nous informer sur sa vulnérabilité. L'objectif est de comparer les techniques d’élastographie ultrasonores endovasculaire (EVE) et non-invasive (NIVE) en fonction de leur potentiel à identifier les composantes calcifiées et lipidiques de la plaque. Les acquisitions intravasculaire et extravasculaire ont été effectuées sur les artères carotidiennes de neuf porcs hypercholestérolémiques à l’aide d’un cathéter de 20 MHz et d'une sonde linéaire de 7.5 MHz, respectivement. Les valeurs de déformation radiale et axiale, rapportés par EVE et NIVE, ont été corrélées avec le pourcentage des zones histologiques calcifiées et lipidiques pour cinq plaques. Nos résultats démontrent une bonne corrélation positive entre les déformations et les composantes calcifiées (r2 = 0.82, P = 0.034 valeur par EVE et r2 = 0.80, P = 0.041 valeur par NIVE). Une forte corrélation entre les déformations axiales et les contenus lipidiques par NIVE (r2 = 0.92, P-value = 0.010) a été obtenue. En conclusion, NIVE et EVE sont des techniques potentielles pour identifier les composants de la plaque et aider les médecins à diagnostiquer précocement les plaques vulnérables. / It is now widely accepted that plaque composition is a major determinant of plaque’s vulnerability to rupture. Since composition of the plaque affects its mechanical properties, the local assessment of mechanical properties of atherosclerotic plaque may inform us about plaque’s vulnerability. The objective is to compare ultrasonic endovascular elastography (EVE) versus non-invasive vascular elastography (NIVE) according to their potential to identify plaque contents. Intravascular and extravascular acquisitions were performed on carotid arteries of nine hypercholesterolemic minipigs with a 20 MHz catheter and a 7.5 MHz standard probe, respectively. Radial and axial strain values, reported by EVE and NIVE respectively, were correlated with histological area of lipid and calcium for five plaques. Our results demonstrate a good positive correlation between strains and calcified contents (r2=0.82, P-value=0.034 by EVE and r2=0.80, P-value= 0.041 by NIVE). Additionally, there is a strong correlation between axial strains and lipid contents by NIVE (r2=0.92, P-value= 0.010). In conclusion, NIVE and EVE are the potential techniques to identify plaque components and to help physicians to early diagnose the vulnerable plaques.