Master of Science / Department of Clinical Sciences / Barret J. Bulmer / Degenerative valve disease is the most common cardiac affliction facing our canine population. To date, canine research has focused on characterizing the disease itself and the histopathological features. Because of the ability to routinely repair or replace diseased valves in human medicine, research focus in humans has been on perfecting these techniques rather than elucidating etiology. The recent interest in valvular interstitial cells has been primarily due to their capacity to degrade collagen with the knowledge that disorganized collagen is a hallmark characteristic of degenerative valve disease. In this project, an easily reproducible cell culture protocol for canine valvular interstitial cells was developed. These cells were phenotyped by utilization of RT-PCR and immunocytochemistry. The use of these cells in a research project looking at response to endothelin exposure with and without protection of vitamin E is demonstrated as an example of the unlimited possibilities for these cells to elucidate not only the etiology of the disease process but also the response to therapy.

Les maladies cardiovasculaires, conséquence de l’athérosclérose, sont la première cause de morbi-mortalité dans le monde et voient leur incidence et sévérité augmenter avec l’expansion de leurs principaux facteurs de risque, tels que l’âge, l’obésité et le diabète. La sténose valvulaire aortique, valvulopathie la plus fréquemment rencontrée dans les pays occidentaux essentiellement chez le sujet vieillissant, partage de fortes similitudes avec l’athérosclérose vasculaire. En effet, les plaques athéroscléreuses et les lésions valvulaires sont le siège de processus d’inflammation, d’angiogenèse, de fibrose et de calcification. Les macrophages, issus de la différenciation tissulaire des monocytes infiltrés, jouent un rôle clé dans l’apparition des lésions athéroscléreuses vasculaires et leur devenir. Leur rôle dans l’état inflammatoire des lésions est aujourd’hui bien établi avec de récentes publications qui font état des propriétés plastiques des macrophages, selon leur microenvironnement. Deux principaux sous types de macrophages ont été décrits dans les plaques athéroscléreuses, les macrophages M1 dit « classiques » et M2 dits « alternatifs ». Leur rôle respectif dans la thrombogénécité, la protéolyse et l’angiogenèse, processus impliqués dans l’instabilité de la plaque, ont été moins étudiés. En revanche, les macrophages sont peu décrits dans la valve, à l’inverse des cellules interstitielles de valves (VIC), qui sont cruciales pour le maintien de l’homéostasie et la fonction valvulaire et sont impliquées dans la fibrose et la rigidité des feuillets valvulaires. Mon travail de thèse a pour objectif d’étudier les rôles des macrophages M1/M2 dans les pathologies vasculaires et valvulaires chez l’homme. Nous nous sommes focalisés sur leurs rôles dans l’instabilité de la plaque athéroscléreuse (processus de coagulation et de remodelage vasculaire) et dans la fibrose valvulaire ainsi que sur leur modulation phénotypique par d’autres types cellulaires présents dans les lésions, les polynucléaires neutrophiles dans la plaque ou les VIC dans la valve.Nos résultats suggèrent que les macrophages M1 et M2 pourraient moduler différemment des processus physiopathologiques majeurs de l'athérosclérose. Par ailleurs, les macrophages M1 de patients diabétiques présentent un phénotype délétère qui pourrait expliquer la plus grande vulnérabilité des plaques d’athérosclérose observée chez ces sujets. Concernant la pathologie valvulaire, après avoir caractérisé par analyse histologique les M1/M2 dans les valves aortiques humaines, nous avons montré que les macrophages M1 sont impliqués dans la progression de la fibrose via la modulation de leur répertoire sécrétoire par les VIC.Cette thèse apporte de nouveaux indices sur les processus physiopathologiques impliqués dans les maladies vasculaires et valvulaires. Elle met l’accent sur le rôle délétère des macrophages M1 chez les sujets diabétiques en pathologie vasculaire et identifie également une fonction jusque-là méconnue des M1 dans la progression de la fibrose, associée au « cross-talk » avec les VIC. Il conviendra par la suite d’identifier les mécanismes moléculaires sous-jacents à ces intéractions, ce qui devrait permettre d’envisager de nouvelles voies thérapeutiques visant à moduler l’effet de ce sous-type cellulaire dans ces pathologies. / Cardiovascular disease, as a result of atherosclerosis, are the main cause of morbidity and mortality in the world and see their incidence and severity increase with the expansion of their major risk factors, such as age, obesity and diabetes. Aortic valve stenosis, valve disease most frequently encountered in Western countries mainly in the old subject, shares strong similarities with vascular atherosclerosis. Indeed, atherosclerotic plaques and valvular lesions are the site of inflammation, angiogenesis, fibrosis and calcification processes. Macrophages, from monocytes infiltrated tissue differentiation, play a key role in the development of vascular atherosclerotic lesions and their future. Their role in the inflammatory state of the lesions is now well established with recent publications that report on plastic properties of macrophages, according to their microenvironment. Two major subtypes of macrophages have been described in the atherosclerotic plaques, classically (M1) or alternatively (M2) activated macrophages. Their respective role in thrombogenicity, proteolysis and angiogenesis processes involved in plaque instability, have been less studied. In contrast, macrophages are not disclosed in the valve, compared to the valvular interstitial cells (VIC), which are crucial for the maintenance of homeostasis and the valvular function and are involved in the fibrosis and rigidity of the valvular leaflets. My thesis aims to study the roles of macrophages M1/M2 in vascular and valvular pathologies in humans. We focused on their roles in the instability of atherosclerotic plaque (haemostatic or clotting process and vascular remodeling) and valvular fibrosis and their phenotypic modulation by other cell types present in the lesions, neutrophils (PNN) in the plaque or VIC in the valve.Our results suggest that the M1 and M2 macrophages may differently modulate major pathophysiological processes of atherosclerosis. In addition, M1 macrophages from diabetic patients have a deleterious phenotype that could explain the increased vulnerability of atherosclerotic plaques observed in these subjects. About valvular pathology, after characterized histologically M1/M2 in human aortic valves, we have shown that the M1 macrophages are involved in the progression of fibrosis through the modulation of their secretory repertoire by VIC.This work provides new clues about the pathophysiological processes involved in vascular and valvular diseases. It focuses on the deleterious role of M1 macrophages in diabetic subjects in vascular pathology and also identifies an unknown function of M1 in the progression of fibrosis associated with "cross-talk" with VIC. It will be necessary later to identify the molecular mechanisms underlying these interactions, which is expected to consider new therapeutic approaches to modulate the effect of this cell subtype in these diseases.

Bisher existiert keine nicht-invasive/ nicht-operative Therapie der Aortenklappenstenose. Als wichtiger Zeitpunkt für eine präventive Maßnahme, zur Verhinderung der Ausbildung einer hömodynamisch relevanten Aortenklappenstenose, kann das Stadium der Aortenklappensklerose angesehen werden. Dieses frühe Erkrankungsstadium verfügt über zahlreiche pathophysiologische Parallelen zur Atherosklerose, für die eine positive Rolle der Prävention durch körperliche Aktivität erwiesen ist. Ziel dieser Arbeit war die Durchführung der Sekundärprävention der kalzifizierenden Aortenklappenerkrankung durch körperliches Training. Um mögliche Effekte dieser Intervention zu eruieren, wurden LDLR-/--Mäuse mit bereits bestehenden pathologischen Aortenklappenveränderungen über einen Zeitraum von 16 Wochen körperlichem Training unterzogen. Durch morphologische, serumanalytische, immunhistochemische und Genexpressionsanalysen konnte abschließend eine Quantifizierung der Effekte körperlichen Trainings - in der Zielsetzung der Sekundärprävention - realisiert werden.

No description available.

No description available.

Abstract Calcific aortic valve disease (CAVD) represents a disease spectrum, ranging from mild aortic valve sclerosis to severe obstructive aortic stenosis (AS), associated with a high risk of myocardial infarction and cardiovascular death. It is a common disease in the Western countries, and with their aging populations, its prevalence is likely to increase. Today, CAVD is recognized as an actively regulated disease. Mechanical stress and endothelial injury are the initiating factors, followed by lipid accumulation and oxidation, leading to inflammation, fibrosis and calcification. Ultimately, the progressive calcification hinders the normal valvular function and obstructs the flow of blood through the valve. The only effective treatment for symptomatic AS is aortic valve replacement. The trials with pharmacological treatments, mainly with anti-atherosclerotic drugs, have not been successful in slowing the progression of the disease. This study was aimed to identify differentially expressed transcripts, and molecular markers taking part in the pathophysiology behind CAVD. In particular, factors related to the renin-angiotensin system, and the apelin – APJ pathway, were investigated during the development of CAVD. In addition, the expressions of granzymes and perforin, as well as podoplanin, were studied in different stages of CAVD. It was demonstrated that these molecules are expressed in aortic valves and dysregulated in AS. These results can help to clarify the mechanisms driving CAVD, thus being potential targets for pharmacological therapy. Furthermore, the studied molecules may reflect the stage and possible subgroups of CAVD. / Tiivistelmä Aorttaläpän ahtauma edustaa tautijatkumoa, joka alkaa lievästä aorttaläpän paksuuntumisesta eli aorttaskleroosista ja jatkuu vaikeaan aorttaläpän kalkkeutuneeseen ahtaumaan eli aorttastenoosiin, johon liittyy korkea sydäninfarktin ja sydän- ja verisuonitatutiperäisen kuoleman riski. Aorttaläpän ahtauma on yleinen tauti länsimaissa, ja väestön ikääntyessä sen esiintyvyys on luultavimmin lisääntymässä. Nykyään aorttaläpän ahtauman tiedetään olevan aktiivisesti säädelty tauti. Mekaaninen rasitus ja endoteelivaurio käynnistävät tautiprosessin, läppäkudokseen kertyy lipidejä ja ne hapettuvat, mikä johtaa tulehdukseen, sidekudoksen lisääntymiseen ja kalkkeutumiseen. Lopulta etenevä kalkkeutuminen heikentää läpän normaalia toimintaa ja estää veren normaalia virtausta sydämestä aorttaan. Ainoa tehokas hoito oireiseen aorttastenoosiin on aorttaläpän korvausleikkaus. Lääkehoitoina on kokeiltu erityisesti ateroskleroosin hoitoon käytettäviä lääkkeitä, mutta niillä ei ole onnistuttu estämään taudin etenemistä. Tässä väitöskirjatyössä tutkittiin molekyylejä ja biokemiallisia reittejä, jotka liittyvät reniini-angiotensiinijärjestelmään ja apeliini-APJ-reittiin. Lisäksi tutkittiin grantsyymien ja perforiinin sekä podoplaniinin ilmentymistä aorttaläpän ahtauman eri kehitysvaiheissa. Tulosten perusteella näitä tekijöitä ilmennetään aorttaläpässä ja niiden määrä on muuttunut kalkkeutuneessa läpässä. Tulokset auttavat osaltaan ymmärtämään aorttaläpän ahtaumaan ja kalkkeutumiseen johtavia mekanismeja, joita voidaan hyödyntää uusia lääkehoidon kohteita suunniteltaessa. Tutkitut molekulaariset tekijät voivat kuvastaa aortan ahtaumataudin vaiheita ja mahdollisia alaryhmiä.

This theses demonstrate practical use of knowledge discovery in the field of cardiac surgery. The tasks of the Department of Cardiac Surgery University Hospital Olomouc are solved through the use of GUHA method and LISp-Miner system. Mitral valve surgery data comes from clinical practice between the years 2002 and 2011. Theoretical part includes chapter on KDD -- type of tasks, methods and methodology and chapter on cardiac surgery -- anatomy and functions of heart, mitral valve disease and diagnostic methods including quantification. Practical part brings solutions of the tasks and whole process is described in the spirit of CRISP-DM.

Introdução: A doença valvar aórtica grave é caracterizada por um processo de acúmulo progressivo de fibrose intersticial no tecido miocárdico. No contexto da sobrecarga mecânica crônica do VE característica dessa condição, a quantidade de fibrose intersticial pode exercer um papel importante na indesejável transição entre hipertrofia ventricular esquerda compensada e insuficiência cardíaca congestiva clinicamente manifesta. Entretanto, a avaliação quantitativa da fibrose intersticial só tem sido possível através da análise histopatológica de fragmentos miocárdicos obtidos por biopsia endomiocárdica. Objetivos: Avaliar se a ressonância magnética (RM) cardíaca com técnica do realce tardio permite a quantificação não-invasiva da fibrose miocárdica quando comparada à análise histopatológica em pacientes portadores de doença valvar aórtica grave. Adicionalmente, avaliou-se a relação entre a quantidade de fibrose miocárdica e parâmetros prognósticos importantes, tais como mortalidade e recuperação funcional do VE após cirurgia de troca valvar aórtica. Métodos: Entre Maio de 2001 e Dezembro de 2003 foram incluídos 54 pacientes com indicação de cirurgia de troca valvar aórtica. Antes da cirurgia, todos os pacientes foram submetidos a RM cardíaca com técnicas de cine-RM e realce tardio miocárdico. A quantificação da fibrose miocárdica pela RM baseou-se na análise das imagens de realce tardio utilizando um novo algoritmo semi-automático. As regiões de fibrose miocárdica foram definidas como o somatório de todos os pixels do tecido miocárdico com intensidade de sinal acima de um limiar definido como: intensidade de sinal média do miocárdio + 2 desvios padrão da intensidade de sinal média da área remota + 2 desvios padrão da intensidade de sinal média do ar. Amostras de tecido miocárdico obtidas por miectomia durante o ato cirúrgico foram submetidas a coloração pelo picrosírius para quantificação da fibrose intersticial. Os pacientes foram submetidos a um segundo exame de RM cardíaca 6 meses após a cirurgia para se avaliar as alterações evolutivas dos parâmetros funcionais do VE e todos foram acompanhados por pelo menos 24 meses quanto à sobrevida após a cirurgia de troca valvar aórtica. Resultados: O percentual de fibrose miocárdica pela RM apresentou boa correlação com os valores obtidos pela histopatologia (r=0,69; y=3,10x+13,0; p<0,0001). A quantidade de fibrose miocárdica, tanto pela histopatologia como pela RM, apresentou correlação inversa significativa com a FE ventricular esquerda basal (r=-0,63 e -0,67 respectivamente; p<0,0001). Adicionalmente, o percentual de fibrose miocárdica apresentou correlação inversa significativa com o grau de recuperação funcional do VE após a cirurgia de troca valvar (r=- 0,42, p=0,04 para a histopatologia; r=-0,47, p=0,02 para a RM). Mais importante, a análise de Kaplan-Meier revelou que o acúmulo de fibrose miocárdica associou-se a menor sobrevida 52±17 meses após a cirurgia de troca valvar (teste log-rank: 2=6,32; p=0,01 para histopatologia; 2=5,85; p=0,02 para RM). Conclusões: A RM cardíaca permite quantificar as regiões de fibrose miocárdica com boa acurácia quando comparada à análise histopatológica nos pacientes portadores de doença valvar aórtica grave. A magnitude de acúmulo de fibrose miocárdica está associada a pior recuperação funcional do VE e a menor sobrevida após a cirurgia de troca valvar aórtica. / Introduction: Severe aortic valve disease is characterized by a process of progressive accumulation of interstitial fibrosis in the myocardial tissue. It has been shown that the amount of interstitial myocardial fibrosis can play an important role in the transition from well-compensated hypertrophy to overt heart failure in the setting of chronic left ventricular mechanical overload typical of this condition. However, assessment of interstitial myocardial fibrosis has only been possible through histological analyses of myocardial fragments obtained from endomyocardial biopsies, which is a complex and invasive procedure and, therefore, with limited clinical applicability. Objectives: Determine whether delayedenhancement cardiac magnetic resonance imaging (MRI) allows for the non-invasive quantification of myocardial fibrosis when compared against histopathological analyses in patients with severe aortic valve disease. Additionally, we evaluated the relationship between the amount of myocardial fibrosis and important prognostic parameters, such as all-cause mortality and LV functional recovery after aortic valve replacement. Methods: Fifty-four patients scheduled to undergo aortic valve replacement surgery were enrolled between May 2001 and December 2003. Before surgery, all patients underwent cine and delayedenhancement MRI in a 1.5 Tesla scanner. Quantification of myocardial fibrosis by cardiac MRI was based on the assessment of the delayed-enhancement dataset using a novel semiautomatic algorithm. The regions of myocardial fibrosis were defined as the sum of pixels with signal intensity above a threshold value defined as: mean signal intensity of the myocardium + 2 standard deviations of mean signal intensity of a remote area + 2 standard deviations of mean signal intensity of air. During open-heart surgery, myectomy samples were acquired from the LV septum and later stained with picrosirius for interstitial myocardial fibrosis quantification. A second cardiac MRI study was performed 6 months after surgery to assess long-term changes in LV functional parameters, and all patients were followed for at least 24 months to evaluate survival after aortic valve replacement. Results: There was a good correlation between the values of myocardial fibrosis measured by MRI and those obtained by histopathological analyses (r=0.69; y=3.10x+13.0; p<0.0001). The amount of myocardial fibrosis, either by MRI or by histopathology, exhibited a significant inverse correlation with LV ejection fraction before surgery (r=-0.63 e -0.67 respectively; p<0.0001). Additionally, the amount of myocardial fibrosis displayed a significant inverse correlation with the degree of LV functional recovery after aortic valve replacement (r=-0.42, p=0.04 for histopathology; r=-0.47, p=0.02 for MRI). Most importantly, Kaplan-Meier and Cox regression analyses revealed that higher degrees of myocardial fibrosis accumulation were associated with worse survival 52±17 months after aortic valve replacement surgery (log-rank test: 2=6.32; p=0.01 for histopathology; 2=5.85; p=0.02 for MRI). Conclusions: Cardiac MRI allows for the non-invasive quantification of myocardial fibrosis with good accuracy when compared with histopathological analyses in patients with severe aortic valve disease. The degree of myocardial fibrosis accumulation is associated with impaired LV functional recovery and worse survival after aortic valve replacement surgery.

Introdução: A doença valvar aórtica grave é caracterizada por um processo de acúmulo progressivo de fibrose intersticial no tecido miocárdico. No contexto da sobrecarga mecânica crônica do VE característica dessa condição, a quantidade de fibrose intersticial pode exercer um papel importante na indesejável transição entre hipertrofia ventricular esquerda compensada e insuficiência cardíaca congestiva clinicamente manifesta. Entretanto, a avaliação quantitativa da fibrose intersticial só tem sido possível através da análise histopatológica de fragmentos miocárdicos obtidos por biopsia endomiocárdica. Objetivos: Avaliar se a ressonância magnética (RM) cardíaca com técnica do realce tardio permite a quantificação não-invasiva da fibrose miocárdica quando comparada à análise histopatológica em pacientes portadores de doença valvar aórtica grave. Adicionalmente, avaliou-se a relação entre a quantidade de fibrose miocárdica e parâmetros prognósticos importantes, tais como mortalidade e recuperação funcional do VE após cirurgia de troca valvar aórtica. Métodos: Entre Maio de 2001 e Dezembro de 2003 foram incluídos 54 pacientes com indicação de cirurgia de troca valvar aórtica. Antes da cirurgia, todos os pacientes foram submetidos a RM cardíaca com técnicas de cine-RM e realce tardio miocárdico. A quantificação da fibrose miocárdica pela RM baseou-se na análise das imagens de realce tardio utilizando um novo algoritmo semi-automático. As regiões de fibrose miocárdica foram definidas como o somatório de todos os pixels do tecido miocárdico com intensidade de sinal acima de um limiar definido como: intensidade de sinal média do miocárdio + 2 desvios padrão da intensidade de sinal média da área remota + 2 desvios padrão da intensidade de sinal média do ar. Amostras de tecido miocárdico obtidas por miectomia durante o ato cirúrgico foram submetidas a coloração pelo picrosírius para quantificação da fibrose intersticial. Os pacientes foram submetidos a um segundo exame de RM cardíaca 6 meses após a cirurgia para se avaliar as alterações evolutivas dos parâmetros funcionais do VE e todos foram acompanhados por pelo menos 24 meses quanto à sobrevida após a cirurgia de troca valvar aórtica. Resultados: O percentual de fibrose miocárdica pela RM apresentou boa correlação com os valores obtidos pela histopatologia (r=0,69; y=3,10x+13,0; p<0,0001). A quantidade de fibrose miocárdica, tanto pela histopatologia como pela RM, apresentou correlação inversa significativa com a FE ventricular esquerda basal (r=-0,63 e -0,67 respectivamente; p<0,0001). Adicionalmente, o percentual de fibrose miocárdica apresentou correlação inversa significativa com o grau de recuperação funcional do VE após a cirurgia de troca valvar (r=- 0,42, p=0,04 para a histopatologia; r=-0,47, p=0,02 para a RM). Mais importante, a análise de Kaplan-Meier revelou que o acúmulo de fibrose miocárdica associou-se a menor sobrevida 52±17 meses após a cirurgia de troca valvar (teste log-rank: 2=6,32; p=0,01 para histopatologia; 2=5,85; p=0,02 para RM). Conclusões: A RM cardíaca permite quantificar as regiões de fibrose miocárdica com boa acurácia quando comparada à análise histopatológica nos pacientes portadores de doença valvar aórtica grave. A magnitude de acúmulo de fibrose miocárdica está associada a pior recuperação funcional do VE e a menor sobrevida após a cirurgia de troca valvar aórtica. / Introduction: Severe aortic valve disease is characterized by a process of progressive accumulation of interstitial fibrosis in the myocardial tissue. It has been shown that the amount of interstitial myocardial fibrosis can play an important role in the transition from well-compensated hypertrophy to overt heart failure in the setting of chronic left ventricular mechanical overload typical of this condition. However, assessment of interstitial myocardial fibrosis has only been possible through histological analyses of myocardial fragments obtained from endomyocardial biopsies, which is a complex and invasive procedure and, therefore, with limited clinical applicability. Objectives: Determine whether delayedenhancement cardiac magnetic resonance imaging (MRI) allows for the non-invasive quantification of myocardial fibrosis when compared against histopathological analyses in patients with severe aortic valve disease. Additionally, we evaluated the relationship between the amount of myocardial fibrosis and important prognostic parameters, such as all-cause mortality and LV functional recovery after aortic valve replacement. Methods: Fifty-four patients scheduled to undergo aortic valve replacement surgery were enrolled between May 2001 and December 2003. Before surgery, all patients underwent cine and delayedenhancement MRI in a 1.5 Tesla scanner. Quantification of myocardial fibrosis by cardiac MRI was based on the assessment of the delayed-enhancement dataset using a novel semiautomatic algorithm. The regions of myocardial fibrosis were defined as the sum of pixels with signal intensity above a threshold value defined as: mean signal intensity of the myocardium + 2 standard deviations of mean signal intensity of a remote area + 2 standard deviations of mean signal intensity of air. During open-heart surgery, myectomy samples were acquired from the LV septum and later stained with picrosirius for interstitial myocardial fibrosis quantification. A second cardiac MRI study was performed 6 months after surgery to assess long-term changes in LV functional parameters, and all patients were followed for at least 24 months to evaluate survival after aortic valve replacement. Results: There was a good correlation between the values of myocardial fibrosis measured by MRI and those obtained by histopathological analyses (r=0.69; y=3.10x+13.0; p<0.0001). The amount of myocardial fibrosis, either by MRI or by histopathology, exhibited a significant inverse correlation with LV ejection fraction before surgery (r=-0.63 e -0.67 respectively; p<0.0001). Additionally, the amount of myocardial fibrosis displayed a significant inverse correlation with the degree of LV functional recovery after aortic valve replacement (r=-0.42, p=0.04 for histopathology; r=-0.47, p=0.02 for MRI). Most importantly, Kaplan-Meier and Cox regression analyses revealed that higher degrees of myocardial fibrosis accumulation were associated with worse survival 52±17 months after aortic valve replacement surgery (log-rank test: 2=6.32; p=0.01 for histopathology; 2=5.85; p=0.02 for MRI). Conclusions: Cardiac MRI allows for the non-invasive quantification of myocardial fibrosis with good accuracy when compared with histopathological analyses in patients with severe aortic valve disease. The degree of myocardial fibrosis accumulation is associated with impaired LV functional recovery and worse survival after aortic valve replacement surgery.