Atherosclerotic plaque (AP) composition is an important factor influencing plaque rupture. Intraplaque hemorrhage (IPH) is a marker of complicated-plaque formation, responsible for many of the clinical manifestations of atherosclerotic disease. Magnetic resonance imaging (MRI) has emerged as a modality to image carotid AP. The in-vivo high-resolution MR imaging of carotid complicated-plaque forms the basis of this thesis. In the first part, Magnetic Resonance Imaging of Intraplaque Hemorrhage (MRIPH), an in-vivo high-spatial-resolution 3-dimensional MRI sequence specifically designed to depict complicated-plaque in the carotid arteries is described. MRIPH was applied, as compared with histologic analysis (gold standard), to demonstrate that T1-hyperintense intraplaque signal represented blood products. Strong agreement was seen between T1-hyperintensity and histologically-identified hemorrhage, with high sensitivity/specificity/positive- and negative-predictive values for T1-hyperintense detection of hemorrhage. While IPH increases plaque rupture risk, high degrees of calcification promote stability. Calcium can generate T1-hyperintensity in some gradient-echo (GRE) sequences. Therefore, distinction between these two components is crucial. In the second part, T1-hyperintensity in MRIPH was shown to be almost exclusively due to hemorrhage and not calcification by directly comparing in-vivo T1-hyperintensity with calcification in ex-vivo specimens imaged with microCT. T1- hyperintesity showed very good albeit inverse agreement with calcification and excellent agreement with lack of calcification as seen on microCT. IPH is thought to be the result of rupture/leakage of the vasa vasora. In the third part, we tested the hypothesis that if IPH were due to vasa vasorum rupture/leakage, the majority of the IPH would be associated with the adventitial rather than the luminal surface of the plaque. Deep (closer to vessel wall) and superficial (closer to vessel lumen) regions of complicated plaques were identified. Very good inter-rater agreement was seen for the location of IPH using MRIPH, with IPH being more frequently present in the deeper compared to superficial segments of the plaque. In summary, an in-vivo MR technique to detect IPH at high spatial-resolution in carotid complicated-plaque was developed; demonstrating T1-hyperintensity in MRIPH is the result of IPH and not calcification. The predilection of IPH for the deeper segments of the plaques suggests that IPH is due to vasa vasorum rupture/leakage.

Atherosclerotic plaque (AP) composition is an important factor influencing plaque rupture. Intraplaque hemorrhage (IPH) is a marker of complicated-plaque formation, responsible for many of the clinical manifestations of atherosclerotic disease. Magnetic resonance imaging (MRI) has emerged as a modality to image carotid AP. The in-vivo high-resolution MR imaging of carotid complicated-plaque forms the basis of this thesis. In the first part, Magnetic Resonance Imaging of Intraplaque Hemorrhage (MRIPH), an in-vivo high-spatial-resolution 3-dimensional MRI sequence specifically designed to depict complicated-plaque in the carotid arteries is described. MRIPH was applied, as compared with histologic analysis (gold standard), to demonstrate that T1-hyperintense intraplaque signal represented blood products. Strong agreement was seen between T1-hyperintensity and histologically-identified hemorrhage, with high sensitivity/specificity/positive- and negative-predictive values for T1-hyperintense detection of hemorrhage. While IPH increases plaque rupture risk, high degrees of calcification promote stability. Calcium can generate T1-hyperintensity in some gradient-echo (GRE) sequences. Therefore, distinction between these two components is crucial. In the second part, T1-hyperintensity in MRIPH was shown to be almost exclusively due to hemorrhage and not calcification by directly comparing in-vivo T1-hyperintensity with calcification in ex-vivo specimens imaged with microCT. T1- hyperintesity showed very good albeit inverse agreement with calcification and excellent agreement with lack of calcification as seen on microCT. IPH is thought to be the result of rupture/leakage of the vasa vasora. In the third part, we tested the hypothesis that if IPH were due to vasa vasorum rupture/leakage, the majority of the IPH would be associated with the adventitial rather than the luminal surface of the plaque. Deep (closer to vessel wall) and superficial (closer to vessel lumen) regions of complicated plaques were identified. Very good inter-rater agreement was seen for the location of IPH using MRIPH, with IPH being more frequently present in the deeper compared to superficial segments of the plaque. In summary, an in-vivo MR technique to detect IPH at high spatial-resolution in carotid complicated-plaque was developed; demonstrating T1-hyperintensity in MRIPH is the result of IPH and not calcification. The predilection of IPH for the deeper segments of the plaques suggests that IPH is due to vasa vasorum rupture/leakage.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13259号 / 論医博第2177号 / 新制||医||1037(附属図書館) / (主査)教授 横出 正之, 教授 富樫 かおり, 教授 湊谷 謙司 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Sur l’île de La Réunion, la prévalence du diabète de type 2 est 3,5 fois plus élevée que celle de la France hexagonale. Parmi les diverses complications qu’engendre le diabète, les AVC qui en résultent sont responsables d’une forte mortalité faisant des maladies cardiovasculaires un problème de santé publique majeur sur l’île. Les AVC ischémiques proviennent de l’occlusion d’une artère cérébrale par un thrombus généré localement ou qui s’est détaché d’une plaque d’athérothrombose généralement localisée au niveau des bifurcations carotidiennes. Les plaques compliquées sont souvent caractérisées par des hémorragies intraplaques, responsables de l’extravasation des cellules sanguines. Différents marqueurs moléculaires, protéiques et physiques peuvent refléter ces processus et renseigner le médecin sur l’instabilité de la plaque du patient. Il est donc important d’étudier ces marqueurs de risque de rupture de plaques carotidiennes chez les patients diabétiques, afin d’en prévenir les complications et la mortalité associée. L’hémorragie intraplaque, notamment pourvoyeuse d’érythrocytes et de neutrophiles libérant leurs contenus cytoplasmiques, participe activement à la déstabilisation de la plaque d’athérothrombose chez le diabétique. Cette thèse a permis l’étude de l’influence de ces marqueurs à travers une étude clinique et de proposer un nouveau concept de phagocytose des érythrocytes glyqués par les cellules endothéliales in vitro. Ainsi, les résultats préliminaires de l’étude clinique nous permettent de supposer que la clairance des globules rouges des patients diabétiques est altérée, ce qui leurs permettraient de résider sur une période plus longue dans les plaques de ces patients. De cette manière, les globules rouges pourraient y être pris en charge par d’autres types cellulaires comme les cellules endothéliales. Nous avons tout d’abord mis au point un modèle de glycation des érythrocytes in vitro reflétant un diabète mal équilibré. Nous avons ensuite mis en évidence que les globules rouges glyqués pouvaient être phagocytés par les cellules endothéliales humaines, conduisant à une prolifération limitée et à la surexpression de l’HO-1. Ces données suggèrent qu’une ingestion des érythrocytes glyqués par les cellules endothéliales pourrait amplifier la déstabilisation des plaques d’athérothrombose carotidiennes des diabétiques. / Type 2 diabetes prevalence in Reunion Island, a French overseas department, is 3.5 higher than in France mainland. Among the various diseases caused by diabetes, stroke induces high mortality making cardiovascular diseases a major public health problem on the island. Ischemic stroke results from a cerebral artery occlusion by a thrombus that is locally produced or has detached from an atherothrombotic plaque usually located at the carotid bifurcations. Complicated plaques can are often characterized by intraplaque hemorrhages, responsible for blood cell extravasation. Several molecular and physical markers can reflect these processes and inform the physician about the instability of the patient's plaque. It is therefore of major importance to study the markers of carotid plaque rupture in diabetic patients in order to prevent complications and associated mortality. Intraplaque hemorrhage, providing erythrocytes and neutrophils releasing their cytoplasmic contents, plays an active role in destabilizing atherothrombotic plaque in diabetic subjects. The objectives of the present thesis were to study these markers in a clinical study and to suggest a new concept of red blood cell phagocytosis by endothelial cells in vitro. According to the first results of the clinical study, we can suggest that in diabetic patients, the clearance of red blood cells is impaired. This prolonged residence of red blood cells in atherosclerotic plaques from diabetic patients. In this way, red blood cells could be phagocytosed by othercell types such as endothelial cells. In this work, we have also set up an in vitro model of erythrocyte glycation that reflects a clinical situation of poorly controlled diabetes. We have demonstrated that glycated red blood cell phagocytosed by human endothelial cells, leading to their limited proliferation and to HO-1 overexpression. These data suggest an ingestion of glycated erythrocytes by endothelial cells may amplify the destabilization of carotid atherothrombotic plaques in diabetics.

L'athérosclérose est une maladie cardiovasculaire complexe affectant la paroi artérielle où le développement et la progression de la plaque sont fortement favorisés par une inflammation chronique. L'instabilité de la plaque carotidienne peut conduire à de potentiels évènements ischémiques majeurs tels que l'accident vasculaire cérébral (AVC) dont le caractère imprévisible rend la prévention primaire très compliquée. Ainsi, il n'existe pas à l'heure actuelle de biomarqueurs prédictifs efficaces de la rupture de plaque. Néanmoins, il est maintenant clairement établi que l'hémorragie intraplaque (IPH), la néovascularisation et l'accumulation excessive de macrophages sont les principaux facteurs d'instabilité de la plaque. Sur la base de travaux précédents, l'objectif de cette thèse était d'évaluer de manière indépendante les effets de l'activité physique (AP) et de la sédentarité, premièrement sur les paramètres histologiques d'instabilité de plaque, et deuxièmement, sur les facteurs de risque secondaires de l'athérosclérose, que sont l'inflammation, le stress oxydant et le profil hémorhéologique de patients asymptomatiques à risque d'AVC traités chirurgicalement. La 1ère étude a montré que l'AP régulière était associée à une prévalence d'IPH diminuée, et était l'unique facteur protecteur de l'IPH. Cette étude a également suggéré un effet bénéfique de l'AP sur le stress oxydant, ainsi que sur l'accumulation de macrophages. Dans une 2ème étude, nous avons caractérisé l'état fonctionnel de protéines potentiellement impliquées dans les dysfonctions du système immunitaire, et l'implication des cellules inflammatoires dans ces mécanismes. Nous avons alors identifié une cytokine pro-inflammatoire jouant un rôle déterminant dans les processus inflammatoires de déstabilisation de plaque. L'étude 3 nous a permis de caractériser l'effet du niveau d'AP sur la réponse monocytaire chez des patients avec plaque d'athérosclérose, et d'identifier une chimiokine qui pourrait avoir un rôle dans la modulation de la réponse monocytaire par l'AP. Enfin, la 4ème étude démontre l'altération de paramètres hémorhéologiques chez des patients atteints de maladie carotidienne sévère, et comment l'AP permet de limiter cette altération via la diminution de l'agrégation érythrocytaire. Ce travail de thèse apporte des informations quant à la pratique de l'AP dans la prévention primaire de l'athérosclérose. Des études complémentaires seront toutefois nécessaires afin de confirmer ces résultats, en proposant notamment une approche interventionnelle en activité physique / Atherosclerosis is a complex cardiovascular disease that affects the arterial wall where plaque development and progression are severely promoted by chronic inflammation. Carotid plaque destabilization could lead to potential major ischemic events such stroke which is still unpredictable, making primary prevention very complex. Thus, there is still currently no suitable predictive biomarker of plaque rupture. Nevertheless, it is now clearly established that intraplaque hemorrhage (IPH), neovascularization and excessive macrophage accumulation are the three main risk factors of plaque instability. Based on previous studies, the aim of this work was to evaluate independently the impact of physical activity (PA) and sedentary behavior, first on histological parameters of plaque instability, and secondly on secondary risk factors of atherosclerosis, such as inflammation, oxidative stress and hemorheological profile of asymptomatic patients at high-risk of stroke who underwent endarterectomy surgery. The first study shows that regular PA was associated to a decreased occurrence of IPH, and was the only protective factor for IPH. This study also suggested a beneficial effect of PA on macrophage accumulation as well as on oxidative stress. Then, in the 2nd study, we have characterized the functional state of proteins potentially implicated in immune system dysfunctions, and the implication of inflammatory cells in these mechanisms. We have identified a pro-inflammatory cytokine as a key driver of disrupting inflammatory process of plaque. In the same way, we have characterized in the 3rd study, the effect of PA on the monocytic response in atherosclerosis patients, and identified a chemokine associated that could explain the modulation of this monocytic response by PA. Finally, the 4th study demonstrates the hemorheological parameters alteration in carotid artery disease patients, and how PA could limit this alteration via red blood cell aggregation. This PhD thesis provided information regarding regular PA in primary prevention of atherosclerosis. However, additional studies are required to confirm these results, using in particular PA interventional approach