Tonic arterial smooth muscle can produce strong contractions indefinitely by formation of slowly cycling crossbridges (latchbridges) that maintain force at a high energy economy. To fully understand the uniqueness of mechanisms regulating tonic arterial contraction, comparisons have been made to phasic visceral smooth muscles that do not sustain high forces. This study explored mechanisms of force maintenance in a phasic artery by comparing KCl-induced contractions in the tonic, femoral artery (FA) and its primary branch, the phasic saphenous artery (SA). KCl rapidly (5 N/m2) and [ca2+]i (250 nM) in FA and SA. By 10 min, [ca2+]i declined to 175 nM in both tissues but stress was sustained in FA (1.3 x 105N/m2) and reduced by 40% in SA (0.8 x l05 N/m2). Reduced tonic stress correlated with reduced myosin light chain (MLC) phosphorylation in SA (28% vs. 42% in FA). SA expressed more MLC phosphatase than FA, and permeabilized (β-escin) SA relaxed more rapidly than FA in the presence of MLC kinase blockade, suggesting that MLC phosphatase activity in SA was greater than that in FA. The reduction in MLC phosphorylation in SA was insufficient to account for reduced tonic force (latchbridge model), and SA expressed more "fast" myosin isoforms than did FA. Cytochalasin-D reduced force-maintenance more in FA than SA. These data support the hypothesis that strong force-maintenance is absent in SA because expressed motor proteins do not support latchbridge formation, and because actin polymerization is not stimulated.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Iveta Rajtmajerová Supervisor: PharmDr. Jana Pourová, Ph.D. Title of diploma thesis: The in vitro effects of selected fenolic substances on isolated rat vessels Silymarin flavonolignans are endowed with various aspects. Recently their hepatoprotective effect has been discussed widely as well as their positive influence on the cardiovascular system. This diploma thesis is concerned with the vasodilatory effects of silybin-A, silybin-B, silybin-A + B and isosilybin-A on rat aorta. The aim of this study was to test the vasodilatory effect of selected substances. The dependence of aortic relaxation on the increasing concentration of tested substances was measured. Isolated Wistar breed rat aorta was used. DRC curves were created and EC50 values were assessed from the obtained values of vessel tension. The results were evaluated subsequently. The vasodilator potential of silybin-A and isosilybin-A is comparable; hence the position of the substituents may be irrelevant in having any impact on relaxation of the vessels. Different vasodilatory effects of silybin-A and silybin-B have been observed thus, the relaxation could be stereoselective. The testing of the equimolar mixture of silybin-A and...

L’anévrisme de l’aorte abdominale (AAA) est une pathologie devant être traitée par un acte chirurgical afin d’éviter une rupture, mortelle dans la majorité des cas. Son traitement par chirurgie endovasculaire (EVAR) a pour objectif d’éviter toute opération trop lourde et invasive en déposant une endoprothèse au sein de l’AAA par voie artérielle, à laquelle le chirurgien accède via une simple incision de l’artère fémorale. Les diffeìrents retours cliniques montrent globalement que les probleÌmes post-opeìratoires observeìs - migration, plicature, endofuite, rupture – sont intimement lieìs aÌ la conception des endoprotheÌses actuelles, reìaliseìes par l’assemblage d’un corps textile avec un exosquelette sutureì sur ce dernier. Ces dernières preìsentent aujourd’hui un comportement mécanique treÌs heìteìrogeÌne, et éloigné de celui de l’aorte native. Ces problèmes post-opératoires conduisent très souvent à une nécessité de ré-opération des patients, ce qui réduit à long terme les bénéfices de la chirurgie endovasculaire.Les sollicitations in-vivo auxquelles est soumise l’endoprothèse sont diverses. La sollicitation principale de gonflement engendrée par la pression du flux sanguin, s’accompagne d’une compression exercée par les organes environnants, mais également de la flexion due aux mouvements du patient et aux géométries tortueuses des anévrismes, etc. Le comportement mécanique de l’endoprothèse doit être biomimétique dans la mesure du possible afin de se calquer sur la portion artérielle d’origine, i.e. limiter des discontinuités mécaniques aux collets, responsables d’hyperplasie intimale.Dans ce contexte, cette thèse a pour objectif de contribuer au développement et à l’optimisation d’endoprothèses vasculaires, issues d’une technologie de fabrication innovante permettant d’insérer une trame hélicoïdale métallique en NiTi au sein d’un corps textile PET tricoté par maille jetée. Cette technologie permet de réaliser des structures textiles homogènes sans exo-squelette. Dans le but d’optimiser les structures produites, différents essais expérimentaux ont été mis en place, couplés à des techniques d’imagerie 2D ou 3D – micro-tomographie RX synchrotron et de laboratoire – afin de caractériser leur comportement mécanique ainsi que leur comportement au fluide (perméabilité). Les résultats de ces différents essais sont comparés lorsque cela est possible à des résultats expérimentaux ou numériques de la littérature sur l’aorte saine, ainsi qu’à des données concernant des produits du marché. Le but final de ces travaux est d’accéder au développement d’une structure endoprothétique type, dont le comportement est proche de celui de l’aorte abdominale native et permettant de limiter les problèmes post-opératoires observés aujourd’hui. / Abdominal aortic aneurysm (AAA) is a pathology that must be repaired by a surgical procedure to avoid a rupture, lethal in most of the cases. Endovascular aneurysm repair (EVAR) is a non-invasive procedure, allowing the deployment of a stent-graft inside the aneurysmal sac. Nevertheless, clinical feedback currently available for stent-grafts show that many complications after surgery – migrations, thrombosis, endoleaks, rupture – are closely linked to the mechanical behaviour of the stent-grafts, which is heterogeneous and very far from the one of the aorta. This behaviour is induced by the design of current stent-grafts, made by assembling a textile body with an exoskeleton sutured on the latter. These post-operative problems frequently lead to a second operation of the patient, which drastically reduces the benefits of endovascular surgery.In-vivo, stent-grafts are submitted to various loadings: inflation induced by the blood pressure, compression due to surrounding organs, but also bending due to the patient motion or the aorta tortuosity, etc. As far as possible, the mechanical behaviour of stent-grafts must also be as closed as possible to the original aorta, to avoid mechanical discontinuities that may be responsible for intimal hyperplasia.In this context, the principal purpose of this thesis is to contribute to the development and the optimization of vascular endoprostheses, resulting from an innovative manufacturing technology whose target is to insert a helical metallic wire in NiTi within a knitted PET textile body. This technology allows to build homogeneous textile tubes without any exo-skeleton. In order to optimize these structures, various experimental tests coupled with 2D or 3D imaging techniques – Synchrotron and laboratory X-ray micro-tomography – have been developed to characterize the mechanical behavior as well as permeability of these tubes. The results of these different tests are compared to experimental or numerical results of the literature regarding the native aortic tissue, as well as data concerning market products. The ultimate goal of this work is to provide new stent-grafts whose behavior is close to the native abdominal aorta, and which limit current post-operative problems.

Este estudo se propôs a avaliar o potencial inflamatório das partículas eliminadas na exaustão do diesel (PED) e 1,2-naftoquinona (1,2-NQ) sobre outros compartimentos, como o músculo liso vascular (aorta torácica; RTA) e do corpo cavernoso isolados de ratos (RCC) e os mecanismos envolvidos via ensaios funcionais e bioquímicos. A injeção i.tr. das PED e 1,2-NQ em ratos Wistar causou inflamação e hiporreatividade das vias aéreas associados ao aumento significativo do relaxamento evocado pela ACh na RTA. No RCC desses mesmos animais, tanto o GTN quanto o estímulo elétrico (EFS) causou maior relaxamento. O conteúdo basal de TBARs na RTA e pulmão foi reduzido, embora outros testes indicadores de estresse oxidativo e / ou atividade antioxidante não mostraram diferenças entre os grupos. As taxas de expressão gênica / protéica da nNOS no RCC de ratos não diferiram do grupo controle, mas a expressão da eNOS e iNOS foi reduzida na RTA e RCC. Não foram quantificadas concentrações séricas do TNF<font face=\"Symbol\">&#945 ou IL-1<font face=\"Symbol\">b, sugerindo que os efeitos sistêmicos ocorrem independentemente destas citocinas. Conclui-se que o tratamento agudo de ratos com a mistura de poluentes induziu inflamação das vias aéreas (e hiporreatividade), capaz de afetar outros compartimentos, como a musculatura lisa vascular (RTA) e do RCC. / We tested the hypothesis that local inflammation in the airways evoked by intra-tracheal instillation of the environmental chemical 1,2-naphthoquinone (1,2-NQ) and diesel exhaust particles (DEP) is capable of targeting other systemic compartments, such as vessels (rat thoracic aorta; RTA) and corpus cavernosum (RCC), and possible involved mechanisms. After 3h, this treatment induced airways hyporresponsiveness to ACh and local inflammation. This effect was associated with decreased numbers of leukocyte in the blood and spleen and increased number of leukocytes in the bone marrow. Pollutant treatment also markedly increased ACh-induced relaxation in RTA and by both GTN- and electrical stimulation-induced relaxation in RCC. Exposure to pollutants did not affect FE-induced contraction in RTA. Neither serum levels of cytokines (TNF<font face=\"Symbol\">&#945 e IL-1<font face=\"Symbol\">b) nor basal concentration of total nitrate were different amongst groups. No evidence of increased catalase activity in RTA, RCC and lung was found. The treatment reduced the eNOS e iNOS gene expression in RTA e RCC, without significantly affecting the nNOS gene expression in RCC. Our results are consistent with the hypothesis that DEP-induced airways hiporresponsiveness and inflammation can account to produce systemic changes, such as structural and functional changes in the RTA and RCC by means of substances derived from endothelium or due to the ability of these pollutants to act to stimulate the production of scavenger of free radical.

Aneurismas são dilatações permanentes iguais ou superiores a 50% do diâmetro original de uma artéria ou do diâmetro proximal da mesma, de acordo com consenso publicado pela Sociedade de Cirurgia Vascular da América do Norte e Sociedade Internacional de Cirurgia Cardiovascular em 1991 (Vliet e Boll, 1997). Aneurisma de aorta é uma doença vascular que afeta mais de 5% da população masculina com mais de 55 anos de idade (How et al., 2005). Desde 1991 um novo método vem sendo utilizado como tratamento deste quadro clínico. Este novo método é considerado menos invasivo e com menor comorbidade, uma vez que se trata da instalação de uma endoprótese por meio de catéter através da artéria femoral. Neste trabalho, será verificado o escoamento através de três aneurismas de aorta abdominal (AAA) e das endopróteses indicadas para o tratamento de cada um dos aneurismas. Foram feitas simulações computacionais afim de se obter as pressões, as velocidades do escoamento sanguíneo e as tensões de cisalhamento na parede das geometrias, usando pulsos de pressão e velocidade fisiológico de um individuo adulto, normotenso, em repouso. No modelo de escoamento simulado, foi adotado o sangue como fluido Newtoniano, incompressível e homogêneo. Foi usado um modelo de turbulência K-, com interpolador upwind de 2ª ordem e tratamento de parede nas geometrias, para que se fosse possível obter resultados satisfatórios (das camadas próximas à parede, e de todos os vórtices). Os resultados obtidos no trabalho foram satisfatórios, uma vez que eles expressaram de forma realista o comportamento do escoamento sanguíneo pelos aneurismas e endopróteses. Foi possível justificar com esse trabalho a instalação de endopróteses em pacientes, quando necessário, com o objetivo de diminuir significativamente a degradação do vaso, causando a ruptura do aneurisma. / Aneurisms are permanently dilatation higher than 50% of the original diameter of an artery or of the proximal diameter of them, according to the published consensus of the Vascular Surgery Society of North America and International Society of Cardiovascular Surgery on 1991 (Vliet and Boll, 1997). Aortas Aneurism is a vascular disease that affects more than 5% of the male population, with more than 55 years old (How et al, 2005). Since 1991, a new method is used in the treatment of these diseases. This new method is less invasive and has less comorbidity. This method is provided by the installation of an endoprosthesis through the catheter through the femoral artery. In this work, the flow will be verified through three abdominal aortic aneurysms (AAA) and the endoprosthesis indicated for the treatment of each of the aneurysms. Computational simulations were made in order to obtain the pressures, velocities of the blood flow and the shear stresses on the wall of geometries, using a pulse pressure and velocity of an individual physiological adult normotensive at rest. For the fluid simulations, it was adopted the blood as Newtonian fluid, incompressible and homogeneous. In simulations, it was used a K- model of turbulence, with 2nd order upwind interpolator and wall geometry treatment in order to obtain satisfied results (on the layers near the walls, and all the vortices). The results obtained in this work were satisfied, once they expressed the realistic behavior of the blood flow through the aneurysm and the endoprosthesis. It was possible to justify with this work the installation of an endoprosthesis inside patients, when it is necessary, in order to decrease significantly the vessels degradation, causing the aneurysm rupture.

A vasculopatia crônica do transplante constitui um dos principais obstáculos para o sucesso do transplante de órgãos a longo prazo. Caracteriza-se pela formação de uma camada neoíntima, que culmina no estreitamento da luz do vaso com consequente isquemia e falência do órgão transplantado. Embora diversos mecanismos imunológicos tenham sido descritos, a patogênese da vasculopatia crônica do transplante ainda não foi esclarecida e continua sem tratamento específico. Neste contexto, estratégias com alvos imunomodulatórios são de extrema importância. Dentre estas possíveis estratégias destaca-se a talidomida, uma droga que apresentada potentes propriedades anti-inflamatórias e imunomodulatórias e que recentemente voltou a ter indicações clínicas. Assim, os objetivos do presente estudo foram: 1) Padronizar o modelo experimental de vasculopatia crônica do transplante no modelo experimental de transplante de aorta, pois este modelo mimetiza as principais características observadas na parede do vaso durante o processo de rejeição; 2) Analisar o efeito da talidomida sobre as alterações morfológicas vasculares (histologia para Verhoeff), componentes celulares (expressão de a-actina, atividade proliferativa, número de macrófagos e linfócitos T, através de imuno-histoquímica), além da participação da apoptose (técnica de TUNEL); 3) Avaliar o potencial efeito anti-inflamatório e imunomodulador da talidomida neste modelo, através da análise da expressão de mediadores da resposta imune (TNF-a, IL-1b, IL-6, IL-2, INF-g, IL-4 e IL-10), bem como a atividade do NFkB e o envolvimento das subunidades p50 e p65 no enxerto vascular. Foram utilizados ratos machos das linhagens isogênicas específicas, Fisher 344 e Lewis, distribuídos em 3 diferentes grupos (32 por grupo): ISO (transplante isogênico de aorta, Fisher para Fisher); ALO (transplante alogênico de aorta, Fisher para Lewis); ALO+TALID (transplante alogênico de aorta tratado com talidomida (200mg/Kg/dia/gavagem)). Os animais foram acompanhados por um período de 30 dias e, em seguida, sacrificados. Os resultados obtidos demonstraram que a padronização do modelo experimental de transplante de aorta foi viabilizada dentro de uma sequência metodológica extremamente precisa e reprodutível. Como esperado, os animais do grupo ISO não apresentaram sinais de rejeição no enxerto vascular, mantendo íntegra a arquitetura das camadas do vaso. Por outro lado, animais do grupo ALO desenvolveram vasculopatia crônica do transplante caracterizada pelo espessamento das camadas íntima e adventícia. Na camada média evidenciou-se diminuição das VSMC e acentuado rompimento das fibras elásticas. Além disso, os animais do grupo ALO apresentaram aumento da expressão de a-actina acompanhada de intensa atividade proliferativa celular, particularmente nas camadas neoíntima e adventícia, provavelmente pela migração das VSMC para a camada íntima. A análise por IH revelou intenso infiltrado de macrófagos, linfócitos T, além de apoptose presente em todas as camadas do aloenxerto. A expressão gênica e a concentração tecidual dos mediadores da resposta imune TNF-a, IL-1b, IL-6, IL-2, INF-g e IL-10 também foram significativamente aumentadas no grupo ALO. Esses achados associaram-se com a ativação do NFkB, bem como o envolvimento dos dímeros p50 e p65. O tratamento com talidomida promoveu um efeito vasculoprotetor através da redução da camada neoíntima e da inflamação local. Além disso, induziu diminuição da expressão gênica e da concentração tecidual dos mediadores TNF-a, IL-1b, IL-6, IL-2, INF-g, bem como aumento da IL-4 e IL-10. A talidomida também diminuiu a ativação do NFkB. Conclusão: os resultados do presente estudo indicam que o modelo experimental de vasculopatia crônica do transplante constitui um modelo adequado para estudar as alterações nas camadas do vaso durante o processo de rejeição ao aloenxerto. A talidomida exerceu um efeito vasculoprotetor, atenuando a formação da neoíntima e do processo inflamatório local, bem como dos mediadores da resposta imune, provavelmente devido as suas propriedades anti-inflamatórias e imunomodulatórias / Chronic transplant vasculopathy is a major obstacle for the long-term success of organ transplantation. It is characterized by the formation of neointimal layer that culminate in the narrowing of the vessel lumen with consequent ischemia and failure of the transplanted organ. Although several immunological mechanisms have been described, the pathogenesis of chronic transplant vasculopathy remains unclear and continues without specific treatment. In this context, immunomodulatory strategies are extremely important. Among these possible strategies stands out thalidomide, a drug that display a potent antiinflammatory and immunomodulatory properties and recently have returned to clinical indications. The aims of this study were: 1) To standardize the experimental model of chronic transplant vasculopathy, induced in an experimental model of aortic transplantation, because this model mimics the key features observed in the vessel wall during the rejection process; 2) To analyze the effects of thalidomide on vascular morphological (Verhoeff staining), cellular components (a-actin expression, proliferative activity, number of macrophages and T lymphocytes by immunohistochemistry), and the participation of apoptosis (TUNEL assay); 3) To evaluate the antiinflammatory and immunomodulatory potential effects of thalidomide in this model by analyzing the mediator expression of the immune response (TNF-a, IL-1b, IL-6, IL-2, INF-g, IL-4 e IL-10), as well as the NFkB activity and involvement of p50 and p65 subunits in the vascular graft. We used male rats from the specific inbred strains, Fisher 344 and Lewis, divided into 3 different groups (32 per group). ISO (isogeneic aortic transplantation, Fisher to Fisher); ALO (allogeneic aortic transplantation, Fisher to Lewis); ALO+THALID (allogeneic aortic transplantation treated with thalidomide (200mg/Kg/day/gavage)). The animals were followed during a period of 30 days and then sacrificed. The results showed that the standardizing of the experimental model of aortic transplantation was possible within a sequence highly accurate and reproducible methodology. As expected, the animals of the ISO showed no sign of graft rejection keeping the entire layered architecture of the vessel. On the other hand, the ALO group developed chronic transplant vasculopathy characterized by thickness of the intima and adventitia. Furthermore, ALO group animals showed increase of a-actin expression accompanied by an intense cellular proliferative activity, particularly in the neointima and adventitia layers, probably due to VSMC migration to the intima. Analysis by IH revealed intense infiltration of macrophages and T lymphocytes, as well as apoptosis in all layers of the allograft. The gene expression and tissue concentrations of the mediators of immune response TNF-a, IL-1b, IL-6, IL-2, INF-g, and IL-10 were also significantly higher in the ALO group. These findings were associated with the NFkB activation, as well as the involvement of p50 and p65 dimers. Treatment with thalidomide promoted a vascular protective effect by reducing the neointimal formation and local inflammation. Moreover, induced a decrease in the gene expression and tissue concentration of the inflammatory mediators TNF-a, IL-1b, IL-6, IL-2, INF-g, as well as increase of IL-4 and IL-10. Thalidomide also decreased the NFkB activation. Conclusion: the results of this study indicate that experimental model of chronic transplant vasculopathy is an appropriate model to study changes in the vessel layers during the process of allograft rejection. Thalidomide promoted a vascular protective effect, attenuating neointimal formation and local inflammation, as well as mediators of immune response, probably due to its anti-inflammatory and immunomodulatory properties

O efeito de contraste espontâneo na aorta torácica foi recentemente associado à progressão da aterosclerose coronariana e aos eventos isquêmicos encefálicos. A casuística estudada foi uma população que fez um ecocardiograma transesofágico constituída de: um grupo caso, 224 pacientes com eventos isquêmicos recentes (<30 dias) e o grupo controle, 85 pacientes com diagnóstico/suspeita de cardiopatia ( 46 foram excluídos), onde avaliamos a possibilidade de uma associação independente entre efeito de contraste espontâneo na aorta e eventos isquêmicos encefálicos recentes, e a associação independente entre vários fatores de risco (id ade~60 anos, histórias de hipertensão arterial sistêmica, de diabete melito, de tabagismo e de disl ipidemia, e da fibrilação atrial) com eventos isquêmicos encefálicos recentes. Avaliamos ainda a associação independente entre diag nósticos ecocardiográficos (efeito de contraste espontâneo no átrio esquerdo, placas complexas aórticas e excrescências de Lambi) - potenciais fatores de risco - e eventos isquêmicos encefálicos recentes. Estudou-se, também, a associação entre efeito de contraste espontâneo na aorta e diagnósticos dos ecocardiogramas transesofágicos (efeito de contraste espontâneo no átrio esquerdo, placas complexas aórticas e excrescências de Lambi), que são potencia is fatores de risco para os eventos isquêmicos encetã licos. Foram excluídos os indivíduos com: diâmetro da aorta transversa e/ou descendente >/4 cm, dissecção da aórtica, fração de encurtamento < 25%, fração de ejeção (Simpson) <40%, aqueles nos quais não houve consenso entre a leitura dos observadores, e os do grupo controle com algum episódio isquêmico sistêmico e/ou encefálico. A metodologia estatística utilizada foi a elaboração de tabelas de contingências, obtendo-se estimativas de risco relativo através da razão de chance com seus respectivos intervalos de confiança de 95% para as análises bivariadas e 90% para as multivariadas. A significância estatística das associações foi determinada através dos testes de Qui-quadrado e exato de Fisher sempre que necessário. As variáveis quantitativas foram comparadas pelo teste 1 de Student. Os efeitos de confusão foram controlados pela análise multivariada através da técnica de regressão logística. Os níveis de signifícância estatística aceitos foram para as análises bivariadas foram a =O,OS e, para as análises multi variadas, a=O, 1. O efeito de contraste espontâneo na aorta mostrou associação com os eventos isquêmicos encefálicos (RC=2,83) e a análise multivariada demonstrou ser esta associação independente (RC=2,03). Além disso, na análise bivariada, vários outros fatores também mostraram associação, sendo que os de maior destaque foram as excrescências de Lambi (RC=6.50), histórias de hipertensão arterial sistêmica (RC=3,79), de diabete melito (RC=2,96), de dislipidemia (RC=2,77), placas complexas aórticas (RC=3,27) e idade </60 anos (RC=2,54). Na análise multivariada, a história de hipertensão arterial sistêmica foi o único fator clínico de risco associado com eventos isquêmicos encefálicos (RC=2,68). Entre os diagnósticos obtidos pelo eco - e que são potenciais fatores de risco -, as placas complexas aórticas (RC=2,92) e as excrescências de Lambi (RC=6,93) foram associadas de maneira independente com os eventos isquêmicos encefálicos. Com relação à associação entre o efeito de contraste espontâneo na aorta e os tàtores de ri sco clínicos e com os diagnósticos ecocardiográficos, a análise bivariada demonstrou que de maior destaque foram o efeito de contraste espontâneo no átrio esquerdo (RC=6,02), a idade ;:::: 60 anos (RC=5,32), a fibrilação atrial (RC=4,8) e as histórias de diabete melito (RC=3,84), de hipertensão arterial sistêmica (RC=2,39), de dislipidemia (RC=2,00) e as placas complexas aórticas (RC=2,17). Na análise multivariada, os fatores mais intensamente associados foram a idade ;:::: 60 anos (RC=5,43), o efeito de contraste espontâneo no átrio esquerdo (RC=4,41 ), as histórias de tabagismo (RC=2,33), de dislipidemia (RC= l ,75), de hipertensão arterial sistêmica (RC=1,67), a fibrilação atrial (RC=3,51) e as excrescências de Lambi (RC=2,06). Em conclusão, a análise destes resultados permite-nos afirmar: 1) que o efeito de contraste espontâneo na a011a torácica está associado de forma independente com os eventos isquêmicos encefálicos recentes; 2) que a história de hipertensão arterial sistêmica foi o único fator de risco que mostrou associação independente com os eventos isquêmicos encefálicos recentes; 3) que as placas complexas aórticas e as excrescências de Lambi apresentaram associação independente com os eventos isquêmicos encefálicos recentes; 4) que a idade 2:: 60 anos, as histórias de hipertensão arterial sistêmica, de tabagismo, de dislipidemia e a fibrilação atrial, foram fatores de risco associados independentemente com o efeito de contraste espontâneo na aorta torácica; 5) a presença de efeito de contraste espontâneo no átrio esquerdo e as excrescências de Lambi foram associadas de maneira independente com o efeito de contraste espontâneo na aorta torácica. / The effect of spontaneous contrast in the thoracic aorta was recently associated to coronary atherosclerosis progression and to encephalic ischemic events. The studied population - consecutive series of patients submitted to a transesophageal echocardiogram - was compounded of two groups: the case group, 224 patients with recent ischemic events (<30 days) and the contr·ol group, 85 patients with diagnosis/suspicion of a heart disease ( 46 were excluded), and the possibility of an independent association between the effect of aortic spontaneous contrast and recent encephalic ischemic events, and the independent association of various risk factors (> 60 years old, anamnesis of systemic arterial hypertension, of diabetes mellitus, of smoking, of dislipidemia and atrial fibrillation) with recent encephalic ischernic events. The independent association between echocardiographic diagnosis ( effect of spontaneous contrast in the letl atrial cavity, cornplexes aortic plaques and strands) - potential risk factors - and recent encephalic ischemic events was also studied. The exclusion criteria was: aortic diameter >4 em, aortic dissection, shortening fraction <25%, ejection fraction <40%, lack of consensus between the readers, and subjects from the control group that suffered a previous systemic/encephalic embolic episode. Statistical methodology utilized was based on the elaboration of contingency tables with relative risk estimation with risk of chances with its respective intervals of95% for the bivariate analysis and 90% for the multivariate analysis. The signifícance of the associations was determincd with the Chi-squarc test and the Fi sher test. The quantitative variables were compared by the Student I test. Multivariate analysis and the logistic regression controlled thc effects of confusion. The levei of statistical significance for the bivariate analysis was a.=0,05 and a.=O.l for the multivariate analysis. The ao11ic efTect of spontaneous contrast exhibited an association with recent ischcmic cncephalic events (RC=2,83) and the multivariate analysis showed that this association is an independent one (RC=2,03). The bivariate analysis other risk factors also showed an association. The most ones being strands (RC=6.50), anamnesis of systemic arterial hypcncnsion (RC=3,79), of diabetes mellitus (RC=2,96), of dislipidemia (RC=2, 77), complexes aortic plaques (RC=3,27) and ~ 60 old (RC=2,54). With the multivariate analysis the anamnesis o f systemic arterial hypertension was the isolated c linicai ri sk làctor that was associated with recent encephal ic event episodes (RC=2,68). The echocardiographic diag nosis - that are potential risk factors - as complexes aortic plaques (RC=2,92) and the strands (RC=6,93) were associatcd m an independent manner with the recent encephalic ischemic events. Regarding the association between the effect of aortic spontaneous contrast and the clinicai risk factors with the echocardiographic results, the bivariate analysis demonstrated that the spontaneous contrast efTect in the le f1 atriurn (RC=6,02), ~ 60 years old (RC=5,32), atrial fibrillati on (RC=4,8) and the anamncsis of diabetes mell itus (RC=3,84), of systemic arterial hypertension (RC=2,39), of dislipidernia (RC=2,00) and the aortic complexes plaques (RC=2, 17) was found . With the multivariate analysis, the risk factors associated were ~ 60 yea rs old (RC=5,43), the efTect of spontaneous contrast in the le ft atrium (RC=4,41 ), the anamnesis o f smoking (RC=2,33), o f disli pidemia (RC= I. 75), o f systemic an crial hypcncnsion (RC= I ,67), atrial fi brillat ion (RC=3,5 I) and strands (RC=2,06). In conclusion, the analysis of our results confirmed that: 1) the spontaneous thoracic aortic effect is independently associated with the recent encephalic ischemic events; 2) the anamnesis of systemic arterial hypertension was the only risk factor that showed an independent association with the recent encephalic ischemic events; 3) the complexes aortic plaques and the strands showed an independent association with the recent encephalíc ischemic events; 4) the age ~ 60 years old, the anamnesís o f systemic arterial hypertension, smoking, dislipidemia and atrial fibrillation, were independently associated risk factors with the effect of spontaneous contrast in the thoracic aorta; 5) the presence of spontaneous contrast in the left atrial cavity and the strands were índependently associated with the effect of spontaneous contrast in the thoracic aorta.

Background: The incidence of coronary heart diseases, including atherosclerosis, increases with ageing. The factors which influence arterial function, and which may be changed with ageing, are multiple but effects of perivascular adipose tissue (PVAT) on large arteries have not previously been considered. A key role for nitric oxide (NO) in mediating the anti-contractile capacity of PVAT has been suggested. Caveolin-1 (Cav-1) modulates the production of NO in vivo by tonic inhibition of eNOS. The influence of aortic PVAT and the contribution of NO to vascular reactivity in ageing C57BL/6 mice, atherosclerotic ApoE knockout mice (ApoE-/-), Cav-1 knockout mice (Cav-1-/-) and atheroprotected ApoECav-1 double knockout mice (ApoE-/-Cav-1-/-) is unknown. Hypothesis: The influence of PVAT on vascular function is modulated by ageing and the development of atherosclerosis via NO bioavailability. Methods: Male mice were used in this study. C57BL/6 mice were obtained at 4 weeks of age and maintained on a normal rodent diet (ND) for 8, 16 or 26 weeks. ApoE-/- and Cav-1-/- mice were bred from in-house colonies and ApoE-/-Cav-1-/- mice were generated by interbreeding ApoE-/- and Cav-1-/- mice. Upon weaning, ApoE-/-, Cav-1-/- and ApoE-/-Cav-1-/- mice were maintained on either a ND or Western-type diet (WD) for 8, 16 or 26 weeks. Vascular reactivity studies on isolated aortic ring preparations were performed in the presence or absence of PVAT. The contribution of NO to the vascular reactivity of aortic PVAT was determined using pharmacological inhibition of NO synthase. Aortic PVAT was assessed for evidence of morphological and/or compositional changes associated with ageing or a WD. Results: NO mediated an anti-contractile effect of aortic PVAT in C57BL/6 mice fed a ND up to 16 weeks. The anti-contractile capacity of aortic PVAT was lost after 26 weeks on a ND and preceded endothelial dysfunction. Loss of the PVAT anti-contractile effect was accompanied by alterations in PVAT morphology and composition. Aortic PVAT from ND-fed ApoE-/- mice was dysfunctional and did not exert an anti-contractile effect. Furthermore, a WD did not alter the influence of PVAT on vascular reactivity in ApoE-/- mice and PVAT morphology and composition was unchanged. NOS inhibition did not alter the contractile responses. The aortic PVAT of ND-fed Cav-1-/- mice did not exert an anti-contractile effect and PVAT composition was unchanged with increasing age. However, after 26 weeks on a WD, aortic PVAT from Cav-1-/- mice potentiated contractions to phenylephrine and white adipocyte hypertrophy was observed. NOS inhibition revealed a pro-contractile effect of aortic PVAT from Cav-1-/- mice. Loss of Cav-1-/- conferred significant protection against the development of atherosclerosis in WD-fed ApoE-/-Cav-1-/- mice despite a proatherogenic lipid profile. Aortic PVAT from ND-fed ApoE-/-Cav-1-/- mice did not exhibit an anti-contractile capacity and PVAT morphology was unchanged with ageing. Additionally, a WD did not influence the effect of PVAT on vascular reactivity in ApoE-/-Cav-1-/- mice although white adipocyte hypertrophy was observed after 26 weeks of high fat feeding. NOS inhibition revealed a pro-contractile effect of aortic PVAT in 8-week ND-fed ApoE-/-Cav-1-/- mice. Conclusions: This work has produced novel insights into the influence of aortic PVAT and NO on vascular reactivity and the morphology of aortic PVAT in ageing C57BL/6 mice, atherosclerotic ApoE-/- mice, Cav-1-/- mice and athero-protected ApoE-/-Cav-1-/- double knockout mice. Ageing to pre-middle age in C57BL/6 mice results in a loss of the anti-contractile effect of PVAT prior to endothelial dysfunction. This is associated with altered NO bioavailability and changes to the morphology and composition of PVAT. This may reveal potential therapeutic targets to restore the anti-contractile capacity of PVAT if comparable age-related PVAT dysfunction is observed in humans. Aortic PVAT of ApoE-/- mice does not exert an anti-contractile effect which may be attributed to decreased basal eNOS activity. A WD does not alter the vascular reactivity of PVAT. In addition, aortic PVAT from Cav-1-/- mice does not exhibit an anti-contractile capacity yet it exerts a pro-contractile effect after 26 weeks on a WD. The aortic PVAT of ApoE-/-Cav-1-/- mice does not modulate vascular reactivity and this is unaltered with feeding of a WD although white adipocyte hypertrophy was observed within the PVAT. The critical role of Cav-1 in the initiation and progression of atherosclerosis is reinforced by the atheroprotected phenotype of the ApoE-/-Cav-1-/- mice even though a severely proatherogenic lipid profile is observed in both the ND and WD-fed mice. Therapeutically targeting LDL transcytosis into the arterial wall could potentially prevent or halt the development of atherosclerosis. Aortic PVAT of ND-fed Cav-1-/- and ApoE-/-Cav-1-/- mice may not be dysfunctional but unable to modulate vascular reactivity due to attenuated vasoconstrictor responses of PVAT-denuded aortic rings as a result of excess NO, although this requires further investigation.

Haematopoietic stem cells (HSC) are invaluable, due to their potential to treat malignant and non-malignant diseases. Modern medicine requires a reliable source of human HSCs (hHSCs) for efficient transplantations, which in many cases cannot be obtained from a single donor. Therefore, the ability to amplify donor hHSCs ex vivo would be an ideal alternative. Past attempts to expand hHSCs in vitro, demonstrated that the protocols developed so far have limited success. My research studied the factors which can affect the optimal culture of transplantable HSCs using a 3D culture system that had previously been used to culture HSCs derived from the aorta-gonad-mesonephros (AGM) region of the mouse embryo. This system involved cell culturing at the gas-liquid interface which is particularly sensitive to mechanical disturbances. To overcome this problem, floating Polypropylene support (rings) were designed and tested and I demonstrated that this was able to prolong aggregate culturing for up to 21 days. Further optimisation tests included altering factors such as oxygen levels, and the presence of antioxidants and apoptosis inhibitors in mouse HSCs culture. I have shown that moderate hypoxia (6% O2) did not affect HSCs in culture, while 2% of O2 led to a significant decrease of HSCs activity. Normoxia resulted in higher reactive oxygen species generation, which would likely be detrimental to cells. However, unexpectedly no improvement in repopulation efficiency of cultured HSCs was achieved by the addition of antioxidant. I also found that when the AGM region was dissociated and co-aggregated in the presence of Rho kinase inhibitor a higher level of repopulation was achieved. In addition, troloxpifitrin-a and p38 inhibitor blocked HSC development without affecting progenitor frequency or the total number of live cells. Subclones of mouse stromal cell line (OP9) were used to create a defined haematopoietic niche for hHSC. Functional screening of these lines in co-aggregate culture re- vealed that 3 of the 34 subclones tested were able to maintain hHSC in culture and repopulate immunodeficient mice at a comparable level to uncultured CD34+ cells. The repopulation in engrafted recipients persisted for over 6 months and showed both myeloid and lymphoid potential. These 3 subclones therefore appeared to create a functional niche for hHSCs and were subsequently used to study the impact of a number of factors including SCF, rock inhibitor, TGFb inhibitor, StemRegenin1 (SR), and prolonged culture technique on hHSC expansion. A significant level of fluctuation between experiments was observed and no definitive conclusions could be drawn. I also attempted to establish stromal cell lines from the human AGM region, more specifically from the ventral (AoV) and dorsal (AoD) regions of the dorsal aorta. Despite attempts to immortalise primary stromal cells, all lines went through a growth crisis. Nevertheless, 30 lines were screened for their ability to support haematopoietic cells in co-aggregate culture with results suggesting that lines derived from AoV expanded haematopoietic precursors more efficiently than AoD lines and OP9 control. Many of the tested lines were able to maintain long-term repopulating human HSCs but the level of repopulation was not as high as that achieved from uncultured CD34+ cells. Unfortunately, these human stromal cell lines have an unstable karyotype which may have an impact on their functional characteristics and they may not represent the nature of the primary cells.

Os stents são dispositivos intravasculares implantados com o objetivo de dilatar ou fixar a placa de colesterol contra a parede arterial. O objetivo avaliar dois tipos de stents de aço inoxidável, um recoberto com poliéster (dacron) e outro não recoberto, implantados na aorta infra-renal de suínos jovens, foram avaliados por morfometria digital para medir o espessamento intimal. Foi realizado um estudo experimental randomizado, separados em dois grupos(stents não revestidos e revestidos com dacron) e duas fases (I e II). Oito stents recobertos com dacron e oito stents de aço inoxidável (30mm de extensão e 8 mm de diâmetro), não revestidos, foram implantados através de abordagem retroperitoneal na aorta infrarenal normal de 16 suínos normolipêmicos. Para a passagem do sistema de implante, foi necessário uma pequena arteriotomia na aorta distal (fase I). Após quatro semanas, a aorta com os stents foram removidas em monoblocos (fase II). Os valores de hematimetria e do lipidograma foram coletados nas duas fases e não apresentaram alterações que pudessem influenciar o estudo. Amostras de tecido dos sítios de fixação (proximal e distal) dos stents foram retiradas, confeccionadas lâminas, que foram coradas pelas técnicas de hematoxilina e eosina de Verhoeff e enviadas para análise morfométrica digital. A camada intima distal não apresentou diferença estatística significativa. A camada média proximal das porções proximal e da porção distal não apresentaram diferença entre os grupos. Os dois tipos de stents apresentaram 100% de perviedade, boa biocompatibilidade e boa incorporação a parede aórtica de suínos normolipêmicos. A camada íntima proximal do grupo de stents revestidos com dacron apresentou espessura maior do que os stents não revestidos, mas com significância estatísitica limítrofe. / Stents are intravascular devices implanted with the objective of dilating or fixing the cholesterol plate against the arterial wall. To evaluate, by digital morphometry, the intimal thickening after the placement of two different kinds of stents: polyester covered stainless steel stents (Dacron) and non-covered stents implanted in young pigs’ infrarenal aortas. The stents were made at the Biotransformation Laboratory of Federal University of Rio Grande do Sul. The experiment was performed at the Biotério of University of Caxias do Sul, carried out on two separate groups of pigs. Eight polyester-covered stainless steel stents (Dacron) and eight stainless steel stents (30-mm long, 8-mm diameter) were deployed through extraperitoneal approach in the normal infrarenal aorta of 16 normolipemic pigs. To allow the passage of the delivery system, a small arteriotomy was performed (phase I). After four weeks, the aorta with the stent was removed en bloc. (phase II). The values of the hematimetric and lipid analysis did not show any changes that could influence the study. Tissue samples of the fixing sites (proximal and distal) of the stents were taken. Microscope slices were prepared and submitted to Verhoeff’s hematoxilin and eosin techniques and sent to morphometric analysis.The intima immediately proximal to the device was thicker in the group of covered stents with marginal significance. The distal intimal layer, proximal medial layer of the proximal portion and distal portion did not present statistical difference between the two groups. Patency was 100% in both groups of these normolipemic pigs. After four weeks the intimal layer immediately proximal to the covered stents was ticker when compared to uncovered stents, but this had a borderline significance. The intimal layer distal to covered stents and the media proximal or distal to the devices had similar morphometric features when covered and uncovered stents where compared.