Dissertations / Theses on the topic 'Intima Hyperplasia'

Die moderne Gefäßchirurgie bedient sich bei hohen Stadien der pAVK, spezieller Gefäßrekonstruktionen in Form von distalen End-zu-Seit-Gefäßanastomosen. Das langfristige Versagen der Gefäßanastomose hängt primär von der Entstehung einer subendothelialen Intimahyperplasie (IH) ab. Diese IH-Gebiete befinden sich je nach Anastomosengeometrie im Gebiet der Hauben- und Fersenzone sowie am Boden der Anastomose. // Mit Hilfe der Particle Image Velocimetry-Technik wird eine Taylor-Patch-, eine Miller-Cuff-Anastomose und eine femoro-crurale Patch-Prothese bezüglich ihrer Flussmuster sowie ihrer hämodynamischen Eigenschaften wie Geschwindigkeit, Scherstress und Rotation in z-Richtung (Vorticity) untersucht. // In einem hydrodynamischen Kreislaufmodell werden elastische, transparente Silikonmodelle der Anastomosen hergestellt und mit einem blutanalogen Newtonschen Fluid (Glycerol-Wasser-Gemisch) unter Simulation der femorocruralen Druckkurve, pulsatil bei Variation der Strömungsbedingung perfundiert. Der periphere Widerstand beträgt 0,5 mmHg/ml/min (PRU) und die Phasenverschiebung -12 Grad (zwischen Druck- und Flusskurve). // Die Flussmuster variieren zwischen den unterschiedlichen Ausstromverhältnissen erheblich. Bei den unterschiedlichen Flussstärken hingegen ähneln sich die Flussmuster. Alle drei Modelle zeigen ausgeprägte Flussseparationszonen im Hauben- und Fersengebiet sowie geometrieabhängig auch eine Stagnationszone am Boden. Diese Bereiche wiesen die geringsten Fluidgeschwindigkeiten, deutlich unter normalem Wandscherstressniveau liegende Scherstressverhältnisse sowie geringe Vorticitywerte auf. Im Bereich der Übergangszonen finden sich hohe Scherstress- sowie Vorticitywerte. Geschwindigkeitsunterschiede des Fluids zeigten sich im Bereich der Ausstromsegmente. Variable Stressverteilungen zeigen sich auch innerhalb der Separationszonen. Eine Erklärung für die unterschiedlich beschriebenen Offenheitsraten der drei Anastomosenformen wird durch diese Arbeit nicht gefunden.<br>Modern vascular surgery uses special termino-lateral anastomoses for treating high levels of peripheral arterial disease (PAD). Long term stenoses and occlusions of vascular anastomoses mostly depend on the development of subendothelial myointimal hyperplasia (MIH). There are characteristic areas within the anastomoses, where this process can be examined: The heel, the tow and the floor zone. // This examination observes local hemodynamics like velocity, shear stress and vorticity (rotation in z-direction) and flow patterns of a Taylor-Patch-, a Miller-Cuff-Anastomosis and a feroro-crural patch prothesis (FCPP) with the usage of a Particle Image Velocimetry. In a hydrodynamic circulation model various elastic, transparent silicon phantoms of termino-lateral anastomoses are perfused with a Newton fluid blood analogon (glycerol-water mixture) while simulating the femorocrural pressure curve in a pulsatile manner under variation of the flow conditions. The outflow resistance is 0.5 mmHg/ml/min (PRU, peripheral resistance units) and a phase shift of -12° between flow and pressure curve is simulated. // The flow patterns differed extremely in accordance of the various outflow ratios. Using different flow intensity, the flow patterns are very similar. // All three anastomoses show characteristic heel and toe separation zones. In the FCPP centre a stagnation zone on the floor can not be examined. Shear stress inside the flow separations was significantly lower than normal wall shear stress. High shear stress levels were found inside the transition zones between flow separation and high velocity mainstream. An explanation for the different stenoses and occlusions time of the three different anastomoses can not be found.

Intimal hyperplasia is associated with graft failure and vascular sutures in the first year after surgery and in postangioplasty restenosis. Allium sativum (common garlic) lowers cholesterol and has antioxidant effects; it also has antiplatelet and antitumor properties and, therefore, has great potential to reduce or inhibit intimal hyperplasia of the arteries. Attempts have been made to inhibit intimal hyperplasia of the arteries with cilostazol and other drugs. Therefore, it is important to address the following research question: what is the difference between the mean of post-angioplasty myointimal thickening in the iliac artery of rabbits treated with A. sativum and that treated with cilostazol? Our objective is to determine the mean difference of post-angioplasty myointimal hyperplasia in the external iliac artery of rabbits with induced atherosclerosis, and compare the outcome between treatment with A. sativum and treatment with cilostazol. This is a randomized preclinical trial study conducted in experimental animals. We used female New Zealand rabbits (Oryctolagus cuniculus), submitted to an atherosclerotic diet and angioplasty of the external iliac artery. The animals were divided into the following groups (n = 10 each) according to treatment: group A, A. sativum, 800 μg⋅kg-1⋅day-1, orally; group C, cilostazol, 50 mg/day, orally; group S, 10 mL of 0.9% physiological saline solution, orally. Our primary variable is the difference of the mean myointimal hyperplasia. The secondary variable is the mean plate thickness of the arterial wall. Complementary data include frequency of limb ischemia, limb loss frequency, frequency of hematoma or bruising, frequency of infection, frequency of bleeding, frequency of animal death, and mean of lipid levels. The sample size was arbitrated in 30 rabbits. Statistical analysis was performed by using ANOVA and Tukey tests, as well as the χ2 test. We calculated the 95% confidence interval (CI) for each point estimate, and the P value was set as < 0.05. Group S had a mean hyperplasia rate of 35.74% (95% CI, 31.76–39.71%); group C, 16.21% (95% CI, 13.36–19.05%); and group A, 21.12% (95% CI, 17.26–25.01%); P < 0.0001. In conclution the Allium sativum quite as effective in inhibiting myointimal hyperplasia compared with those treated with cilostazol.<br>A hiperplasia intimal está relacionada à falha de enxertos e suturas vasculares no primeiro ano pós-cirurgia e nas reestenoses pós-angioplastia. O Allium sativum (alho comum) reduz o colesterol e tem efeito antioxidante, antiplaquetário e antitumoral; logo havia grande possibilidade de reduzir ou inibir a hiperplasia da íntima das artérias, a qual sua inibição vem sendo tentada com o Cilostazol e outros tratamentos. Sendo assim é relevante responder a pergunta de pesquisa: qual a diferença de média de hiperplasia miointimal pós-angioplastia na artéria ilíaca de coelhos tratados com Allium sativum comparada aos tratados com Cilostazol? O objetivo é determinar a diferença de média de hiperplasia miointimal pós-angioplastia na artéria ilíaca externa de coelhos com aterosclerose induzida e tratados com Allium sativum comparada aos tratados com Cilostazol. O estudo foi um ensaio pré-clínico aleatório em animais de experimentação por 35 dias. Os animais foram coelhos fêmeas (Oryctolagus cuniculus), da linhagem Nova Zelandia, submetidos à dieta ateroslcerótica e à angioplastia da artéria ilíaca externa direita. Os animais foram divididos em: Grupo A (n=10) coelhos tratados com a Allium sativum (800 μg/kg/dia), em doses diárias, por via oral. Grupo C (n=10): coelhos tratados com Cilostazol em doses diárias de 50 mg/dia, por via oral. Grupo S (n= 10) coelhos tratados com 10 mL de soro fisiológico 0,9%, por via oral, que foi nosso controle negativo. A variável primária foi a diferença de frequência da média de hiperplasia miointimal. Variáveis secundárias: a média de espessura da placa na parede arterial. Dados complementares: a frequência isquemia do membro, a frequência de perda do membro, a frequência de hematoma ou equimose, a frequência de infecção, a frequência de sangramento, a frequência de morte do animal e a média dos níveis lipídicos. O tamanho da amostra foi arbitrado em 30 coelhos. A análise estatística foi realizada com o teste ANOVA, Qui-quadrado e Tukey. Foi calculado o intervalo de confiança de 95% para cada ponto estimado. Os resultados foram: grupo S teve um índice médio de hiperplasia de 35,74% IC de 95% (31,76% a 39,71%); grupo C teve um índice médio de hiperplasia de 16,21% IC de 95% (13,36% a 19,05%); grupo A teve um índice médio de hiperplasia de 21,12% IC de 95% (17,26% a 25,01%); com P < 0,0001. Sendo assim, o Allium sativum tem a mesma eficácia na inibição da hiperplasia miointimal comparada aos tratados com Cilostazol

Saphenous vein graft disease represents an unresolved problem in coronary artery bypass grafting. After surgery, progressive modification in the vein wall occurs, leading to occlusion of the graft lumen. This process, called intima hyperplasia, involves the participation of vein-resident cells as well as the recruitment of vein-extrinsic cells. Arterial wall strain has recently emerged as one of the factor that can contribute to the pathogenesis of the vein graft disease. Therefore, in collaboration with Department of Bioengineering, Politecnico di Milano we developed a culture system for the ex vivo pressure stimulation of vein segments. This new ex vivo vein culture system is able to reproduce the wall strain typical of the arterial circulation. The ex vivo vein culture system (ECVS) adopted in this project has been validated and proved as a valuable, reliable, easy handling and versatile tool for studying arterial pressure events triggered in VGD. The biological data achieved confirm an important contribution of the arterial-like wall strain in SV structural and biochemical changes, activation of vessel resident cells and in the expression of molecular signals involved in the pathogenesis of IH. Using this system, we found that either venous- or arterial-specific pressure regimens induced vein pro-pathologic commitment involving upregulation of Matrix Metallo-Proteases 2/9, and induction of microRNAs-21/146a/221. By contrast, arterial-like pressure caused a significant morphological rearrangement of the vein, a suppression of Tissue Inhibitor of Metallo Protease-1, an enhanced expression of TGF-β1 and BMP-2 mRNAs and, finally, the upregulation of microRNAs-138/200b/200c. In coronary-pressure stimulated vessels, the density of the adventitial vasa vasorum was significantly increased. This was accompanied by an increased presence of cells co-expressing NG2, CD44 and SM22α markers in the adventitia, identifying them as multipotent mesenchymal cells/smooth muscle cells progenitors with a pericyte origin. An increase in Histone H3 Lysine 4 methylation and histone H4 Lysines 9/16 acetylation levels was finally found in adventitial cells and vasa vasorum. The present findings suggest a mechanistic role of the arterial-like pulsatile pressure in reinforcement of SV-resident cells pro-pathologic commitment in vein bypass failure, by activation of mechanical-dependent transcriptional circuitries and of pericyte-derived cells located in the vessel adventitia. The ultimate goal of this project is to find a treatment that can prevent, avoid or reduce the incidence of the vein graft disease in patients subjected to bypass surgery with saphenous vein. This treatment could include one or more targets identified in this work focusing on the early stage of the pathological adaptation of the SV to the new hemodynamic environment.

Introduction: 12,000 infrainguinal bypass grafts are performed annually in the UK. Despite improvements in surgical technique, outcomes remain suboptimal: 20% of above knee grafts require intervention to maintain patency by 3 years. Only antiplatelet agents have been demonstrated thus far to improve graft survival. 80% of graft failure is as a result of intimal hyperplasia, an inflammatory process characterised by the proliferation and migration of vascular smooth muscle cells. Toll Like Receptors (TLR), part of the innate immune system, have been implicated in atherosclerosis formation but not investigated in a model of infrainguinal graft failure. When a vein is used as a conduit for infrainguinal bypass graft it has been exposed to ischaemic and hypoxic conditions: preliminary data has demonstrated that ischaemic vascular smooth muscle cell explants are hyperproliferative. Phospholipase C γ (PLC γ) is a signalling pathway with potential links to innate immune pathways and pathways induced by hypoxia and ischaemia. Methods: Human vein tissue was obtained from patients undergoing amputation and coronary artery bypass surgery and used for immunohistochemistry and to obtain vascular smooth muscle cells by explant method. Immunohistochemistry was used to determine the presence of TLR4 and PLC γ in human vein tissue. Specific TLR Ligands were used to determine the functional response of TLR’s in vascular smooth muscle cells as measured by Interleukin 8 ELISA. Radiolabelled Thymidine incorporation was used to measure proliferation of vascular smooth muscle cells in response to TLR4 activation, hypoxia and PLC γ inhibition. Results: TLR4 was demonstrated to be present in human vein tissue, and functionally active in human vascular smooth muscle cells. Furthermore stimulation with the specific ligand of TLR4 caused enhanced proliferation of vascular smooth muscle cells. Hypoxia (5% and 10% Oxygen) significantly enhanced proliferative responses of vascular smooth muscle cells. PLC γ was demonstrated to be present in human vein tissue, and inhibition, using U73122 in vascular smooth muscle cells reduced proliferation. Conclusion: TLR activation and hypoxia appear to enhance the proliferative responses of human vascular smooth muscle cells, a key cellular pathway of intimal hyperplasia formation and infrainguinal graft failure. Inhibition of PLC γ reduces proliferative responses. Further research is required to confirm that PLC γ is a key common pathway mediating enhances of proliferation caused by TLR activation and hypoxia.

The most common cause of vein bypass graft failure in the postoperative period of 1 month to 1 year is stenosis, which occurs in up to 30% of arterial reconstructions. This thesis investigates the intimal hyperplasia underlying such lesions using a laboratory model. The first chapter reviews the current literature regarding vein graft stenoses and is followed in Chapter 2 by a brief introduction to tissue and organ culture and their usefulness as investigative research tools. Before embarking on a study of a pathological condition, Chapter 3 studies the structure of the "normal" long saphenous vein in patients undergoing arterial surgery. A degree of intimal thickening was identified in the majority of the veins in this population, the possible causes of which are discussed. The fourth chapter describes and validates an organ culture of human saphenous vein to study the vascular biology of vein graft intimal hyperplasia. Since smooth muscle cell proliferation is a pivotal event in the development of such lesions, a reliable and reproducible method of assessing proliferation was required and is described in Chapter 5. Chapter 6 investigates the effect of endothelial denudation on the development of intimal thickening, and an organ coculture study described in Chapter 7 positively identifies a soluble paracrine mediator produced by the endothelium which can promote intimal hyperplasia. The following chapters utilise variations of the coculture method to further define the precise role played by the endothelium. Chapter 8 demonstrates that isolated, cultured endothelial cells do not promote intimal thickening in denuded veins, suggesting that the normal anatomical location of endothelial cells overlying smooth muscle cells in the vein wall may be important. Chapter 9 therefore describes the development of a method to reseed endothelial cells onto denuded vein segments in order to observe whether the development of intimal hyperplasia can be restored. This proved not to be the case, possibly because the process of culturing had phenotypically altered the endothelial cells, thereby rendering them incapable of producing their paracrine factor. However, a number of other hypotheses and methods by which they could be investigated, are also discussed. The main drawback of human saphenous vein organ culture is that it is a no-flow system. There is considerable evidence in the literature to show that haemodynamics modify the normal and pathological structure and function of blood vessels. Chapter 10 therefore describes the development of an in vitro flow model of saphenous vein graft intimal hyperplasia in an attempt to model the in vivo situation more closely. The final chapter summarises the data presented in this thesis, draws conclusions, and examines prospects for future research in this field.

Cardiovascular diseases (CVD) remain the most common cause of death in the United States. Additionally, peripheral artery disease affects thousands of people each year. A major underlying cause of these diseases is the occlusion of the coronary or peripheral arteries due to arteriosclerosis. To overcome this, a number of vascular interventions have been developed including angioplasty, stenting, endarterectomies and bypass grafts. Although all of these methods are capable of restoring blood flow to the distal organ after occlusion, they are all plagued by unacceptably high restenosis rates. While the biological reactions that occur as a result of each of these methods differ, the initiating factor of both the primary atherosclerosis and subsequent failure of vascular interventions appears to be intimal hyperplasia (IH). Intimal hyperplasia is most simply defined as the expansion of multiple layers of cells internally to the internal elastic lamina of the blood vessel. This excessive cellular growth leads to arterial stenosis, plaque formation and inflammatory reactions. Despite extensive research the underlying factors that cause IH remain unclear. A quantity of research to date has implicated endothelial cell mechanosensation as the mechanism by which IH is initiated with evidence positively correlating wall shear stress with IH. Others, however, have demonstrated that changes in the stresses applied to the wall in vitro can modulate IH independent of hemodynamic shear stress. Thus, relations between wall tensile stress and IH in vivo may shed light on the underlying mechanisms of IH. Since noninvasive measurement of wall tensile stress in vivo is difficult, it is most feasible to measure oscillatory wall strain which is intimately related to wall tensile stress through the mechanical properties of the arterial wall. In this dissertation, we hypothesize that reductions in oscillatory wall strain precede the formation of intimal hyperplasia in a murine model. To test our hypothesis, we first developed a novel, high spatial and temporal resolution method to measure oscillatory wall strains in the murine common carotid artery. We validated this method both in vitro using an arterial phantom and in vivo using a murine model of abdominal aortic aneurysms. To assess relationships between strain and IH, we applied our strain measurement technique to a recently developed mouse model of IH. In this model, a suture is used to create a focal stenosis and reduce flow through the common carotid artery by 85%; resulting in proximal IH formation. Using this approach, we identified a relationship between oscillatory strain reductions and IH. Subsequent analysis demonstrated that early reductions in mechanical strain just 4 days after focal stenosis creation correlate with IH formation nearly 1 month later. Since IH is not expected to form by day 4 in this model, we went on to assess changes in gross vascular morphology at day 4. We discovered that, although strains are significantly reduced by day 4, no significant IH can be observed, suggesting that changes in wall structure are resulting in strain reductions. At day 4 post-op, we observed cellular proliferation and leukocyte recruitment to the wall without intimal hyperplasia. These studies suggest that early reductions in mechanical strain may be an important predictor of IH formation. Clinically, this relation could be important for the development of novel techniques for predicting IH formation before it becomes hemodynamically significant.

This thesis examines aspects of intimal hyperplasia in infrainguinal bypass grafts. There are 3 introductory chapters. Chapter 1 describes the aetiology and treatment of lower limb vascular disease, with particular reference to atherosclerosis. Chapter 2 is a review of the literature regarding the vascular biology of intimal hyperplasia. Chapter 3 is a review of the techniques of cell and organ culture used in the study of vascular disorders. Chapter 4 is composed of four clinical studies of infrainguinal grafts performed at the Leicester Royal Infirmary. There is a prospective study of the long-term benefit of vein graft surveillance. This is followed by a study of the long-term results of percutaneous transluminal angioplasty of vein graft stenoses. A retrospective review of the outcome of polytetrafluoroethylene (PTFE) grafts used in lower limb bypass surgery forms the 3rd part of the chapter. This is followed by a prospective study of the benefits of graft surveillance for synthetic grafts. The next 3 chapters are laboratory based. Chapter 5 looks at the effect of various growth factors on the proliferation of human saphenous vein smooth muscle cells, the ceils involved in the intimal hyperplasia of vein graft stenoses. Chapter 6 looks at aspects of platelet- derived growth factor on the proliferation seen in an organ culture model of human saphenous vein. Chapter 7 examines 2 different ways of incorporating flow in an organ culture of human saphenous vein and compares the effects of high and low shear rates on the development of intimal hyperplasia in the model. In the final chapter, the experimental results are summarised and possible future work relating to the thesis is discussed.

Restenosis due to intimal hyperplasia is a major clinical problem that compromises the success of angioplasty and endovascular surgery. The pathogenesis of restenosis is multifactorial, involving such events as endothelial injury, inflammation, platelet activation, and hyperplasia of the intima, primarily due to vascular smooth muscle cell replication (vSMC). The incidence of intimal hyperplasia varies in different risk populations, e.g., diabetic patients, up to 35% of whom require bare metal stent implantation; clinical evidence has shown that this value is reduced but continues to cause problems after the implantation of drug-eluting stents (DES). Overall, however, despite many years of clinical experience with drug-eluting balloons (DEB), the number of large, high-quality, randomised clinical trials is low, and further data are urgently needed across the spectrum of clinical indications. Taxol and other cytostatic drugs destroy a cell's ability to use its cytoskeleton in a flexible manner, and, considering the clinical results, further research on a more physiologic mechanism of action should be pursued. Antioxidants are currently under investigation due to their protective activity within the vessels. Rosenbaum et al. showed that the endothelialisation of prosthetic grafts was significantly reduced, and anastomotic hyperplasia, significantly increased in rabbits on a high cholesterol diet. Treatment with an antioxidant improves endothelial cell coverage, decreases intimal hyperplasia and reduces oxidative stress, promoting the patency of prosthetic grafts. Resveratrol is a polyphenolic phytoalexinic antioxidant that is produced by grapes and other plants in response to injurious infections. The molecular structure of RSV, unfortunately, reduces its immediate clinical application for three main reasons: 1) its status as a highly lipophilic molecule; 2) its fast drifting from the TRANS- to CIS-phase, representing an oxidised and inactive state, respectively; and 3) its low circadian bioavailability for rapid hepatic metabolism. As consequence of these features, the oral bioavailability of RSV is negligible since it is rapidly conjugated to improve the solubility of the compound. The disposition of 14C-labeled RSV, as orally and intravenously administered in normal, healthy volunteers, was evaluated to estimate the extent of the oral dose absorbed, the bioavailability of the unchanged drug, and the drug’s metabolic phase. RSV demonstrated a high oral absorption but a rapid and extensive metabolism, resulting in only trace amounts of unchanged RSV remaining until systemic circulation. Five major metabolites were detected in the urine samples14, plasma and colo-rectal cancer tissues, although all were only measured qualitatively due to a lack of available reference materials. Metabolite 1 (M1) was a RSV monoglucuronide, and metabolite 2, an isomeric RSV monoglucuronide (M2), was found in greater abundance. M3 was a dihydroresveratrol monoglucuronide, whereas M4 and M5 were two poorly resolved RSV sulphates, i.e., a resveratrol monosulphate (M4) and a dihydroresveratrol sulphate (M5). Despite the controversial results on the efficacy of RSV reported in the literature, very recent data obtained in colon cancer cells have supported the notion that RSV, in spite of its low bioavailability, is able to act through its metabolites, mainly the sulpho-conjugate but also the combination of sulphate/glucuronide derivatives. Despite the wide literature on RSV, only few preclinical studies have demonstrated the efficacy of RSV in an animal model or investigated the possibility of locally administering this antioxidant by eluting stents. Based on our experience, we decided to set up a sterile, injectable, and hydrophilic RSV-containing compound (RSV-c). This solution was locally administered in the common iliac artery of adult male New Zeeland white rabbits using a dedicated device.

Intimal hyperplasia (IH) is characteristic of a cell population increase within the innermost layer of the arterial wall. It is hypothesized that extracellular matrix vascular remodeling secondary vascular injury is dependent upon the Th17 subset of the CD4+ lymphocytes. Male C57BL/6J and FVB/NJ murine strains underwent complete left common carotid artery ligation for periods of 14 and 28 days. A therapeutic simvastatin model was carried out in the FVB/NJ strain and involved a daily subcutaneous injection regimen of 40 mg/kg/mouse beginning 72 hours prior to and daily following a 14 day carotid ligation period. Histological and RT-PCR analysis was carried out with harvested carotid artery samples. The FVB/NJ 14 day and 28 day histological stains of the left common carotid artery following ligation injury developed evident structured and disassembled intimal hyperplasia, respectively. A gene array demonstrated dramatic expression of immune and cytokine transcription markers particularly in the FVB/NJ strain at both ligation time points. IL-17 and IL-6 transcriptional gene expression was upregulated greater than 20-fold in the FVB/NJ 28 day injury model. IL-17 transcription was significantly expressed by a change of 50.06 ± 0.19 (p = 0.004) in this strain at 28 days versus the control. Lastly, the simvastatin treatment model was found to exacerbate the immune response to ligation injury. These results revealed that the immune system elicits a role in the vascular remodeling that potentiates intimal hyperplasia.

Introdução: A reestenose pós tratamento endovascular de lesões ateroscleróticas em artérias periféricas é a principal desvantagem desta técnica minimamente invasiva. A inflamação vascular após angioplastia com balão e/ou implante de stent desempenha um papel importante na proliferação de células do músculo liso vascular e posterior crescimento de uma neoíntima, e vários marcadores inflamatórios têm sido referidos como potenciais preditores dessa complicação, porém os fatores que contribuem para a estenose intra-stent no segmento vascular periférico não foram completamente elucidados. Recentemente, tem-se sugerido que a superfície revestida de stents recobertos possa impedir a reestenose de forma mais eficaz do que os stents convencionais. Objetivo: Avaliar o papel do sistema calicreína-cinina (SCC), do óxido nítrico (NO) e das metaloproteinases (MMP), mediadores inflamatórios importantes e que contribuem ativamente para a reparação de tecidos, no processo de reestenose arterial devido a hiperplasia intimal, pós angioplastia com stent recoberto no segmento fêmoro-poplíteo, com a intenção de contribuir com novas medidas terapêuticas. Método: Foi realizado um estudo prospectivo envolvendo 27 pacientes submetidos à angioplastia com stent revestido no segmento fêmoro-poplíteo, selecionados no Ambulatório de Cirurgia Vascular e Endovascular do HCFMRP/USP. Foram estudados os seguintes marcadores: sistema calicreína-cininas - com quantificação dos substratos (cininogênio de alto e baixo peso molecular - CAPM / CBPM) e da atividade das enzimas (calicreína plasmática e tecidual e cininase II); a determinação dos níveis de nitrito e nitratos para a avaliação de óxido nítrico; dosagem das MMPs 2 e 9 e dos níveis circulantes de seus inibidores (inibidores teciduais das metaloproteinases - TIMPs [1 e 2]). Amostras de sangue foram coletadas antes do implante do stent, 24 horas e seis meses após o procedimento. Foi realizado ultrasson Doppler após seis meses, e, na presença de alterações, realizada angiografia para comprovação da presença de reestenose. Resultados: Quatro (14,8%) dos vinte sete pacientes estudados desenvolveram reestenose (>= 50%) em seis meses. Esses pacientes tiveram níveis significativamente mais baixos de CAPM (24h, P <0,05) e de CBPM (antes - P <0,05; 24 horas P <0,01; 6 meses P <0,05); níveis mais baixos de TIMP 2 ( seis meses P<0,05) comparados ao grupo sem reestenose. As atividades da calicreína plasmática e tecidual, da cininase II, NO e MMPs tiveram comportamento semelhante entre os pacientes com e sem reestenose. Conclusão: As taxas de reestenose foram baixas com o uso de stents revestidos no segmento fêmoro-poplíteo comparativamente aos índices publicados de stents não revestidos. Os pacientes que desenvolveram reestenose mostraram níveis reduzidos de cininogênios e de TIMP-2 (seis meses após a angioplastia). Por outro lado, não foi possível demonstrar a participação do óxido nítrico e das metaloproteinases no processo de reestenose<br>Background: Restenosis after endovascular treatment of atherosclerotic lesions in the peripheral circulation is the major drawback of this minimally invasive technique. Vascular inflammation after balloon angioplasty or stent implantation plays an important role in smooth muscle cells proliferation and subsequent neointima growth, and various inflammatory markers have been reported as potential predictors of this complication, but the factors that contribute to the in-stent stenosis in peripheral vascular segment have not been fully elucidated. Recently, it has been suggested that the coated surface of stents grafts can prevent restenosis more effectively than conventional stents. Objective: The aim of this study was to evaluate the role of the kallikrein-kinin system (KKS), nitric oxide (NO) and metalloproteinases (MMPs), wich are important inflammatory mediators and actively contribute to tissue repair, in the process of arterial restenosis due to intimal hyperplasia, with the aim of developing new interventions. Method: Single-center prospective study with 27 patients with peripheral artery disease (PAD) requiring percutaneous transluminal angioplasty (PTA) and stenting, in the femoropopliteal segment, using coated stents grafts, was performed. The following markers were studied: kallikreinkinin system using the quantification of proteins (high and low weight Molecular kininogen HMWK / LMWK), verification of enzyme activity (tissue kallikrein, plasma kallikrein and kininase II), determination of nitrite and nitrates levels for evaluation of nitric oxide, MMPs 2 and 9 circulating levels and their inhibitors (tissue inhibitors of metalloproteinases [TIMPs 1 and 2]). Serum samples were collected before stent implantation, 24 h and six months after the procedure. Doppler ultrasound was performed after six months, and in the presence of any changes, an angiography was performed to prove the presence of restenosis. Results: Four (14,8%) of the treated patients developed restenosis (>50%) within 6 months. These patients had significantly lower levels of HMWK (24 hours, P < .05), LMWK (before - P < .05; 24 hours - P < .01; 6 months - P < .05) and lower levels of TIMP 2 (6 months < .05) compered to no restenosis group. The activities of plasma and tissue kallikrein, kininase II, NO and MMP had similar behavior among patients with and without restenosis. Conclusion: Restenosis rates were low with the use of coated stents in the femoropopliteal segment compared to published bare metal stents results. Patients with restenosis showed reduced levels of kininogens and TIMP-2 (six months after angioplasty) in patients who developed restenosis. Moreover, it was not possible to demonstrate the involvement of nitric oxide and metalloproteinases in the restenosis process

L’utilisation de greffons artériels (Artère Thoracique Interne (Ati)) et/ou veineux est habituelle au cours de pontages coronaires.1) L’hyperplasie intimale (HI) d’une veine est un mécanisme adaptatif lorsque la veine est soumise à un flux artériel. Certaines solutions de conservation semblent limiter cette HI dans des études in vitro. 2) Deux types de circulation extra-corporelle (CEC) sont utilisables : CEC centrifuge dite non pulsée et CEC à galets dite pulsée. L’absence de pulsatilité est à l’origine d’une réaction inflammatoire systémique et créée une altération du tonus myogénique d’artères intermédiaires chez le rat. L’objectif de ce travail était :1) comparer les solutés de conservation sur des veines implantées en position aortique chez le rat. 2) évaluer l’impact des flux générés par les pompes de CEC sur la fonctionnalité endothéliale des ATIs et la réaction inflammatoire systémique. 1) L’implantation d’un greffon veineux conservé dans du sérum hépariné ou du sang autologue hépariné ou dans une solution anti-oxydante type GALA était à l’origine d’une hyperplasie intimale entrainant une occlusion du greffon dans plus de 50% des cas. Une réaction inflammatoire pariétale importante était retrouvée au niveau de la veine et de l’aorte abdominale distale. 2) La réactivité vasculaire était conservée quel que soit le type de pompes. Une réponse inflammatoire systémique est observée alors que le niveau d’inflammation n’était pas modifié dans les artères. En conclusion, 1) l’utilisation d’une solution anti-oxydante ne permet pas de réduire significativement le risque d’hyperplasie intimale lorsque le greffon veineux est implanté en position artérielle 2) l’exposition d’une ATi à une pompe centrifuge et à galets ne modifie pas la réactivité vasculaire ni la fonctionnalité endothéliale<br>Internal thoracic arteries (ITAs) and venous grafts are commonly used in coronary artery bypass grafting. 1) Intimal hyperplasia (IH) constitutes an adaptative mechanism observed when saphenous vein graft is exposed to arterial blood pressure. Storage solutions aiming on decreasing this IH have been identified in previous in vitro studies. 2) Cardio pulmonary bypass can be performed using either a centrifugal pump, with a non -pulsatile flow or a roller pump, producing a pulsatile flow. Non pulsatile flow was showed to induce a systemic inflammatory response therefor damaging the myogenic tone of resistance arteries in a rat model. The aims of this study were : 1) To compare the impact of storage solutions on saphenous vein grafts implanted in aortic position in a rat model. 2) To evaluate the impact of the flow generated by pumps on endothelial functionality of ITAs and the systemic inflammatory response. 1) IH and graft thrombosis occurred in more than 50% of cases when venous graft was stored in heparinized saline solution, autologous heparinized blood or GALA anti-oxydant solution, and implanted in arterial position. A parietal inflammatory response was observed in venous graft as well as distal abdominal aorta. 2) Vascular reactivity was conserved whatever the pump used. Systemic inflammatory response was observed while the intraparietal inflammatory was not modified in the graft. To conclude, 1) the use of GALA anti-oxydant solution did not decrease the risk of intimal hyperplasia when venous graft is exposed to arterial blood flow 2) exposure of internal thoracic artery to centrifugal and roller pumps did not affect endothelial functionality nor vascular reactivity

This thesis looks at clinical and biological aspects of vein graft stenoses in order to improve on existing management strategies and to explore the possibility for a new pharmacological therapy using antagonists of the endothelin system. After an overview of peripheral vascular disease, the introductory chapters discuss vein graft surveillance, intimal hyperplasia and properties of the vasoactive peptide, endothelin. The work described consists of clinical and laboratory based research. In the clinical chapters a retrospective study analysed the influence of patient factors on the outcome of lower limb vein grafting in the current era of postoperative vein graft surveillance. Following this, two prospective studies examined specific aspects of graft surveillance. Firstly, the predictive value of pre-discharge duplex vein graft scans was determined. The second study validated the criteria for intervention in duplex detected vein graft flow abnormalities. The first laboratory experiments set out to determine the effect of endothelin and endothelin receptor antagonists on proliferation in isolated venous smooth muscle cells. Following this, an organ culture system, a more representative model of intimal hyperplasia, was used to demonstrate the association between endothelin production and development of intimal hyperplasia. Using the same model, a series of experiments were then performed to determine the effect of endothelin inhibition. Endothelin was inhibited at the level of its synthesis, and by none selective and then selective receptor blockade. The final chapter summarises and concludes the main findings and discusses areas of future work that could arise from this thesis.

Includes abstract.<br>Includes bibliographical references (leaves 100-111).<br>This study examines the histopathological response to injury following both balloon angioplasty and endovascular stenting in the Zucker rat, a model that allows interpretation of the role of obesity as well as progressive glucose intolerance and hyperinsulimaemia. Lean and obese Zucker fatty rats and Zucker diabetic fatty rat (ZDF) were subjected to balloon injury with or without stenting. The development of IH, along with the histological response to injury was analyzed.

O objetivo do presente trabalho foi avaliar a morfologia e a ultraestrutura da hiperplasia intimal arterial decorrente de um modelo de estenose aórtica experimental, através da inserção de um pino de acrílico no orifício da artéria renal esquerda. Realizamos um estudo morfológico e ultraestrutural em 64 ratos Wistar, machos, com peso médio de 250 g, divididos em 4 subgrupos de acordo com o dia da eutanásia (1, 7, 15 e 30 dias de pós-operatório). O segmento da aorta envolvendo o pino foi retirado e estudado com diferentes protocolos para: microscopia óptica de alta resolução, microscopia eletrônica de transmissão e de varredura. Uma trombose se formou ao redor do pino 24 horas após a cirurgia, mostrando sinais de organização com 7 dias. Com 15 dias, uma hiperplasia intimal adjacente à base do pino foi visualizada. Esta alteração permaneceu com 30 dias de pós-operatório. Este espessamento era caracterizado principalmente por células musculares lisas provenientes da camada muscular média. A obstrução gera alterações hemodinâmicas locais com repercussão sobre as células endoteliais localizadas próximas à base do pino. Esses achados são discutidos.<br>Objective: To study the light and electron microscopy of intimal hyperplasia induced by experimental aortic stenosis after insertion of a plug into the aorta through the left renal artery. Methods: Sixty-four Wistar male rats, weighing an average of 250g, were allocated into two groups: group control, sham-operated, and group experimental, operated. The animals were killed on days 1, 7, 15, and 30 after surgery. The fragments of aorta implicating the plug were excised and studied using high resolution light microcopy and transmission and scanning electron microscopy. Results and Conclusions: A thrombus was observed around the plug 24 hours after surgery, organized at day 7. An intimal hyperplasia could be observed closed to the basis of the plug 15 and 30 days after surgery. The intimal thickening detected was mainly composed of smooth muscle cells migrated from the medial layer of the aorta intermixed with extracellular matrix. Moreover, the endothelial cells around the plug lost their orientation. Theses findings are discussed.

Introdução: Um dos fatores mais comuns de falha do procedimento das angioplastias vasculares periféricas é a estenose recorrente (reestenose). A inflamação vascular após angioplastia e implante de stent desempenha papel importante na proliferação de células do músculo liso vascular e posterior crescimento de neoíntima hipertrófica. Diversos estudos têm sugerido que a superfície dos stents, quando impregnada com determinadas moléculas, pode limitar a reestenose de forma eficaz. O carbono diamante, polímero de hidrocarboneto amorfo, pode ser usado para essa finalidade, reduzindo liberação de íons metálicos e a trombogenicidade. Diversos mediadores têm sido implicados na resposta inflamatória vascular, como o sistema calicreína-cinina (SCC), as citocinas, proteína C reativa (PCR) e o óxido nítrico (NO). Portanto, estudos clínicos específicos correlacionando o implante do stent aos níveis séricos destes possíveis marcadores podem contribuir no entendimento desse processo. Objetivo: Estudar a perviedade do dispositivo e a evolução clínica de doentes submetidos a angioplastia com stents impregandos de carbono no território vascular periférico, assim como o comportamento de mediadores séricos envolvidos nas fases mais precoces do processo inflamatório pós angioplastia. População e Método: Estudo prospectivo envolvendo 32 pacientes submetidos à angioplastia com stent no segmento ilíacofêmoro-poplíteo, selecionados no Ambulatório de Cirurgia Vascular e Endovascular do HCFMRP/USP. Foram tratadas lesões estenosantes ou oclusivas com angioplastia e colocação de stents de nitinol, com superfície impregnada de carbono (Carbostent®). Foram estudados os seguintes marcadores: SCC - com quantificação dos substratos (cininogênio de alto e baixo peso molecular - CAPM / CBPM) e das enzimas calicreína plasmática e tecidual, além da quantificação da cininase II; a determinação dos níveis de nitrito e nitratos para a avaliação de óxido nítrico; dosagem sérica de PCR e citocinas (IL-1 beta, IL-6, IL-8, IL-10, TNF-alfa e TGFbeta). Também foi realizado a dosagem dos leucócitos. Amostras séricas foram coletadas antes, 24 horas e 6 meses após o implante do stent. Os pacientes foram seguidos por um ano, para avaliação da perviedade nesse período. Resultados: Dos 27 pacientes que completaram seis meses de estudo, houve apenas uma reestenose (3,7%) e nenhuma oclusão (96,3% de perviedade). No período de um ano, quatro pacientes perderam seguimento e todos os 23 doentes avaliados, mantiveram a perviedade dos stents, com exceção do paciente no qual houve reestenose no tempo seis meses. Houve redução significativa das concentrações das citocinas inflamatórias (IL-1 beta, IL-6, IL-8 e TNF-?) no tempo 24 horas e seis meses quando comparado com o pré-procedimento (p<0,05); exceto a IL-8 no tempo 24 horas. As citocinas anti-inflamatórias (IL10 e TGF-beta) comportaram-se de maneira antagônica, com elevação do TGF-beta no tempo 24 horas e elevação, tanto do TGF-beta quanto da IL-10, no tempo 6 meses versus pré-tratamento e 6 meses versus 24 horas (P<0,05). A PCR apresentou queda no tempo seis meses em relação ao tempo 24h (p<0,05). Os níveis de NO não se alteraram entre os tempos; os de leucócitos elevaram-se no tempo 24 horas e reduziram-se no tempo seis meses em relação ao tempo 24 horas (p<0,05). Conclusão: No presente estudo a taxa de reestenose precoce foi de 3,7% nos primeiros 6 meses e 5% em 12 meses de seguimento. O comportamento dos marcadores inflamatórios evidenciou sua correlação direta com o processo de angioplastia e implante de carbostent, em especial o SCC, as citocinas, PCR e os leucócitos. Contudo, não foi possível relacionar a variação dos seus níveis séricos com o processo de reestenose.<br>Background: One of the most common factors of failure of the peripheral vascular angioplasty procedure is recurrent stenosis (restenosis). Vascular inflammation following angioplasty and stent implantation plays an important role in vascular smooth muscle cell proliferation and subsequent hypertrophic neointimal growth. Several studies have suggested that the surface of stents, when impregnated with certain molecules, can limit restenosis effectively. Diamond carbon, amorphous hydrocarbon polymer, can be used for this purpose, reducing release of metal ions and thrombogenicity. Several mediators have been implicated in the vascular inflammatory response, such as the kallikrein-kinin system (SCC), cytokines, C-reactive protein (CRP) and nitric oxide (NO). Therefore, specific clinical studies correlating stent implantation with serum levels of these possible markers may contribute to the understanding of this process. Objective: To study the patency of the device and the clinical evolution of patients undergoing angioplasty with carbon-impregnated stents in the peripheral vascular territory, as well as the behavior of serum mediators involved in the earlier stages of the inflammatory process after angioplasty. Population and Method: Prospective study involving 32 patients submitted to angioplasty with stent in the iliac-femoro-popliteal segment, selected at the HCFMRP / USP Outpatient Vascular and Endovascular Outpatient Clinic. Stenosis or occlusive lesions were treated with angioplasty and placement of nitinol stents with a carbon impregnated surface (Carbostent®). The following markers were studied: SCC - with quantification of substrates (high and low molecular weight cincinogens - HMWC / LMWC) and plasma and tissue kallikrein enzymes, besides quantification of kininase II; determination of nitrite and nitrate levels for the evaluation of nitric oxide; serum levels of CRP and cytokines (IL-1 beta, IL-6, IL-8, IL-10, TNF-a and TGF-b). Leucocytes were also dosed. Serum samples were collected before, 24 hours and 6 months after stent implantation. The patients were followed for one year to assess the patency in this period. Results: Of the 27 patients who completed six months of study, there was only one restenosis (3.7%) and no occlusion (96.3% of patency). In one year, four patients lost follow-up and all 23 patients evaluated, maintained stent patency, with the exception of the patient who had restenosis over time for six months. There was a significant reduction in the concentrations of inflammatory cytokines (IL-1 b, IL-6, IL-8 and TNF-a) in the time 24 hours and six months when compared with the pre-procedure (p <0.05); except for IL-8 in the 24 hour time. Anti-inflammatory cytokines (IL10 and TGF-b) behaved in an antagonistic manner, with TGF-b elevation in the 24 hour time and elevation of both TGF-b and IL-10, at 6 months versus pre- treatment and 6 months versus 24 hours (P <0.05). CRP showed a decrease in time six months in relation to time 24h (p <0.05). NO levels did not change between the times; the leukocytes increased in time 24 hours and reduced in time six months in relation to time 24 hours (p <0.05). Conclusion: In the present study, the rate of early restenosis was 3.7% in the first 6 months and 5% in 12 months of follow-up. The behavior of the inflammatory markers showed its direct correlation with the angioplasty and carbostent implantation process, especially SCC, cytokines, CRP and leukocytes. However, it was not possible to relate the variation of their serum levels to the restenosis process.

Atherosclerosis, the leading cause of mortality in Western societies, affects large elastic arteries, causing focal deposition of proliferative inflammatory and lipid-laden cells within the artery. Several risk factors have been causally implicated in the ???reaction to injury??? hypothesis first described by Ross in 1969. The ???injury??? sustained by endothelial cells may be either mechanical or chemical. Environmental factors have a role in the production of chemical agents that are injurious to the endothelium. Mechanical stresses such as wall tensile stress are proportional to systemic blood pressure and pulse pressure. Essentially, these systemic pressures are fairly evenly distributed throughout the circulation. However, atherosclerotic lesions characteristically occur at focal sites within the human vasculature; at or near bifurcations, within the ostia of branch arteries and at regions of marked or complex curvature, where local haemodynamic abnormalities occur. The most discussed haemodynamic factor seems to be low or highly oscillating wall shear stress which exists on the outer wall of bifurcations and on the inner aspect of curving vessels. The magnitude of these haemodynamic forces may not be great but the subtleties of their variable spatial distribution may help to explain the multifocal distribution of atherosclerotic plaques. With the altered haemodynamics there is endothelial injury and phenotypic changes in the endothelium result, which in turn lead to endothelial cell dysfunction. These haemodynamic variables are difficult to measure directly in vivo. In this work a novel model is developed utilising human autologous vein bypass grafts as a surrogate vessel for the observation of pathological structural changes in response to altered haemodynamics. The influence of haemodynamic factors (such as wall shear stress) in the remodeling of the vein graft wall and the pathogenesis of Myointimal Hyperplasia (MIH) and resultant wall thickening in femoral bypass grafts is analysed. The haemodynamic determinants of MIH (which have been established in many animal models) are similar to those implicated in atherosclerosis. The accelerated responses of the vein (Intimal hyperplasia develops much more rapidly than atherosclerotic lesions in native vessels) make it an ideal model to expediently examine the hypothesised relationships prospectively in an in vivo setting. Furthermore, the utilisation of in vivo data acquired from non-invasive diagnostic methods (such as Magnetic Resonance Angiography (MRA) and Duplex ultrasound) combined with the application of state-of-the-art Computational Fluid Dynamic (CFD) techniques makes the model essentially non-invasive. The following hypotheses are examined: 1) regions of Low shear and High tensile stress should develop disproportionately greater wall thickening, 2) regions of greater oscillatory blood flow should develop greater wall thickening, and 3) regions of lower wall shear should undergo inward (or negative) remodelling and result in a reduction in vessel calibre. The conclusions reached are that abnormal haemodynamic forces, namely low Time-averaged Wall Shear Stress, are associated with subsequent wall thickening. These positive findings have great relevance to the understanding of vein graft MIH and atherosclerosis. It was also evident that with non-invasive data and CFD techniques, some of the important haemodynamic factors are realistically quantifiable (albeit indirectly). The detection of parameters known to be causal in the development of graft intimal hyperplasia or other vascular pathology may improve ability to predict clinical problems. From a surgical perspective this might be employed to facilitate selection of at-risk grafts for more focused postoperative surveillance and reintervention. On a broader stage the utilisation of such analyses may be useful in predicting individuals at greater risk of developing atherosclerotic deposits, disease progression, and the likelihood of clinical events such as heart attack, stroke and threat of limb loss.

As metaloproteinases têm sido implicadas no desenvolvimento de hiperplasias intimais em diferentes situações. O objetivo do presente trabalho foi avaliar a participação das metaloproteinases 2 e 9 no desenvolvimento da hiperplasia intimal decorrente de modelo experimental de estenose intrínseca da aorta. Este modelo consiste na inserção de um pino acrílico na aorta de ratos. Foram utilizados 288 ratos Wistar machos, com peso médio de 250 g, os quais foram separados em quatro grupos: (1) controle sham-operado, (2) controle sham-operado tratado com doxiciclina, (3) estenosado e (4) estenosado tratado com doxiciclina. Estes animais foram tratados com doxiciclina (inibidor não-seletivo de metaloproteinases) 30 mg/Kg/dia e sacrificados nos períodos de 1, 7, e 15 dias após a cirurgia. O segmento da aorta envolvendo o pino foi retirado e estudado com diferentes protocolos para: microscopia óptica de alta resolução e convencional, imunoistoquímica, Western blot para eNOS e iNOS, zimografia convencional e in situ. Um trombo se formou ao redor do pino 24 horas após a cirurgia, mostrando sinais de organização com 7 dias. Com 15 dias, uma hiperplasia intimal adjacente à base do pino foi visualizada. Este espessamento foi caracterizado principalmente por células musculares lisas provenientes da camada muscular média. Apesar das metaloproteinases 2 e 9 estarem inibidas pela doxiciclina (comprovado pela imunoistoquímica, zimografia convencional e in situ), não observamos alterações importantes na composição e desenvolvimento da hiperplasia intimal decorrente deste modelo experimental de estenose intrínseca da aorta<br>Metaloproteinases has been implied in the development of intimal hyperplasia in different situations. We have used an experimental model of aorta stenosis, with a mushroom plug that promotes local turbulence and thrombosis. Methods: 288 Wistar male rats, weighing an average of 250g, were allocated into four groups: control groups, sham-operated and sham-operated treated with doxycycline, and experimental groups, operated and operates treated with doxycycline. The animals were killed on days 1, 7, and 15 after surgery. The fragments of aorta implicating the plug were harvested and studied using high resolution light microcopy, immunohistochemistry, Western Blot to eNOS and iNOS, conventional and in situ zymography. With animal survival of more than 24 h, we followed the partial fibrinolysis of the thrombus as well as its posterior organization and incorporation to the arterial wall as a neointima for up to 15 days. The intimal thickening detected was mainly composed of smooth muscle cells migrated from the medial layer of the aorta intermixed with extracellular matrix. Although, the MMP-2 and 9 were inhibiting, the intimal hyperplasia did not show difference in your composition and development.

Fundamentos: Ensaios prévios reunindo pacientes em series consecutivas ou randomicas sem cegamento evidenciaram beneficio da adição do cilostozol à terapia antiplaquetária em diabéticos submetidos ao implante de stents coronários farmacológicos com redução nas taxas de reestenose binária, perda tardia intra-stent e revascularização tardia da lesão alvo. Objetivos: O objetivo primário deste estudo foi verificar se a adição do cilostazol à dupla terapia antiplaquetária, proporcionaria uma redução adicional da hiperplasia intimal em diabéticos após o implante de stent farmacológico, mensurada por meio do cálculo do volume de obstrução pelo ultrassom intracoronário 9 meses após o procedimento índice. Os objetivos secundários foram aferir a angiografia quantitativa do vaso alvo e ocorrência de eventos cardíacos adversos graves (óbito, infarto do miocárdio não fatal e necessidade de nova revascularização da lesão-alvo) aos 30 dias, 9 meses e 1 ano. Casuística e métodos: Estudo prospectivo, unicêntrico, randomizado, duplo cego, reunindo 133 pacientes diabéticos, comparando pacientes que receberam cilostazol (Grupo 1, n= 65 ) versus placebo (Grupo 2, n= 68), submetidos a implante de stent coronário com liberação de zotarolimus em artéria coronária nativa com estenose maior ou igual a 50% e diâmetro de referência igual ou superior a 2,0 mm (avaliação visual), com reestudo angiográfico e análise ultrassonográfica aos 9 meses. Resultados: Os 2 grupos foram similares nas características clínicas, angiográficas e técnicas, exceto na evidencia de maior incidência de hipertensão arterial no grupo 2 (81,5% vs 94,1%, p=0,026) assim como nos diâmetros dos stents coronários utilizados, significativamente menores no grupo 1 (2,78 mm vs 2,96 mm, p<0,001). O calculo do volume de obstrução intimal por meio do ultrassom intracoronário aos 9 meses foi similar entre os grupos (33,2% vs 35,1%, p=0,069), assim como as taxas de eventos cardíacos adversos graves (12,3% vs 8,8%, p= 0,811), trombose de stent (1,5% versus 0,75%, p= 0,237), reestenose binária intra-sent (9,8% vs 6,8%, p= 0,988), perda tardia intra-stent (0,60 vs 0,64, p=0,300) e no segmento ( 0,57 vs 0,58, p= 0,387). Conclusões: A adição do cilostazol à dupla terapia antiplaquetária com ácido acetilsalicílico e clopidogrel em pacientes diabéticos submetidos à implante de stent com zotarolimus, não reduziu eventos cardíacos adversos graves ou o porcentual de hiperplasia intimal intra-stent mensurado pela análise volumétrica do ultrassom intracoronário.<br>Background: Previous trials with assembled patients in consecutive or random series without blindness offered evidence of the benefit adding cilostazol to the antiplatelet therapy in diabetic patients undergoing drug-eluting stents coronary implantation, with reduction in binary restenosis rates, in-stent late loss and late target lesion revascularization. Objectives: The primary objective of this study was to determine whether the addition of cilostazol to the dual antiplatelet therapy would provide an additional intimal hyperplasia reduction in diabetic patients after drug-eluting stents implantation, measured by calculating the obstruction volume through the intravascular ultrasound 9 months after the index procedure. Secondary objectives were to assess the target vessel quantitative angiography and the occurrence of serious adverse cardiac events (death, nonfatal myocardial infarction and need for a target lesion revascularization) at 30 days, 9 months and 1 year. Methods: Prospective, single center, randomized, double blinded study, gathering 133 diabetic patients, comparing who received cilostazol (Group 1, n= 65) versus placebo (Group 2, n= 68), undergoing coronary stenting, with the releasing of zotarolimus in a native coronary artery with stenosis greater than or equal to 50% and reference diameter equal to or greater than 2.0 mm (visual assessment) with the intravascularultrasound and angiographic restudy at 9 months. Results: Both groups were similar in clinical, angiographic and technical characteristics, except for a higher incidence of arterial hypertension in group 2 (81,5% vs 94,1%, p=0,026) as well as significantly lower coronary stents diameters in group 1 (2,78 mm vs 2,96 mm, p<0,001). The intimal obstruction volume calculated by the intravascularultrasound at 9 months was similar between the groups (33,2% vs 35,1%, p=0,069), as well as the rates of major adverse cardiac events (12,3% vs 8,8%, p= 0,811), stent thrombosis (1,5% versus 0,75%, p= 0,237), in-stent binary restenosis (9,8% vs 6,8%, p= 0,988), in stent late loss (0,60 vs 0,64, p=0,300) and at the segment ( 0,57 vs 0,58, p= 0,387). Conclusions: The addition of cilostazol to the dual antiplatelet therapy with acetylsalicylate acid and clopidogrel, in diabetic patients undergoing stent implantation with zotarolimus did not reduce major adverse cardiac events nor the percentage of intra-stent intimal hyperplasia measured by the intravascularultrasound volumetric analysis.

Die Restenose stellt ein zentrales Problem der interventionellen Kardiologie dar und ist häufigste Komplikation nach perkutanen Angioplastieverfahren. Hauptursache dieser Wiederverengung des Gefäßes ist die Bildung einer Neointima durch die Proliferation transdifferenzierter vaskulärer glatter Muskelzellen und die Sekretion extrazellulärer Matrix. Die Entstehung reaktiver Sauerstoffspezies (ROS) und die Entzündungsreaktion nach der Gefäßverletzung werden als frühe, die Neointimabildung induzierende Prozesse diskutiert. Im Rahmen dieser Arbeit wurden mehrere Projekte bearbeitet, die Aufschluss über die während der Neointimabildung statt findenden Prozesse geben sollen. Mit Hilfe eines Verletzungsmodells der murinen Femoralarterie wurde der Einfluss der Entzündung und der ROS-Bildung auf die Neointimabildung in der Maus untersucht. Die Behandlung mit dem mitochondrialen Superoxiddismutase-Mimetikum MitoTEMPO verminderte die Bildung der Neointima besser, als die Behandlung mit dem globalen ROS-Fänger N-Acetylcystein. Die stärkste Hemmung der Neointimabildung wurde jedoch durch die Immunsuppression mit Rapamycin erreicht. Interferon-γ (INFγ) ist ein wichtiges Zytokin der Th1-Immunantwort, das in Folge der Gefäßverletzung freigesetzt wird und die proinflammatorischen Chemokine CXCL9 (MIG, Monokine Induced by INF), CXCL10 (IP-10, INF inducible Protein of 10 kDa) und CXCL11 (I-TAC, Interferon inducible T cell-Chemoattractant) induziert. CXCL9, CXCL10 und CXCL11 sind Liganden des CXC-Chemokinrezeptors 3 (CXCR3) und locken chemotaktisch CXCR3 positive Entzündungszellen zum Ort der Gefäßverletzung. Daher wurde die spezielle Bedeutung des Chemokins CXCL10 in der Restenose untersucht. Dazu wurden CXCL10-defiziente Mäuse dem Femoralisverletzungsmodell unterzogen und die Gefäße nach 14 Tagen morphometrisch und immunhistologisch untersucht. CXCL10-Defizienz führte in Mäusen zu einer verminderten Neointimabildung, die mit einer verringerten Inflammation, Apoptose und Proliferation im verletzten Gefäß korrelierte. Neben der Inflammation beeinflusst aber auch die Reendothelialisierung der verletzten Gefäßwand die Restenose. Interessanterweise war im Vergleich zu Wildtyp-Mäusen in den CXCL10-Knockout-Mäusen auch die Reendothelialisierung erheblich verbessert. Offensichtlich ist das CXCR3-Chemokinsystem also in völlig unterschiedliche biologische Prozesse involviert und beeinflusst nicht nur die Bildung der Neoimtima durch die Förderung der Entzündung, sondern auch die Unterdrückung der Reendothelialisierung der verletzten Gefäßwand. Tatsächlich wird der CXCR3 nicht nur auf Entzündungszellen, sondern auch auf Endothelzellen exprimiert. Zur separaten Untersuchung der Rolle des CXCR3 in der Inflammation und der Reendothelialisierung wurde im Rahmen dieser Arbeit damit begonnen konditionelle CXCR3-Knockout-Mäuse zu generieren, in denen der CXCR3 entweder in Entzündungszellen oder in Endothelzellen ausgeschaltet ist. Zum besseren Verständnis der molekularen Mechanismen, mit denen der CXCR3 seine Funktionen vermittelt, wurde zudem untersucht ob dieser mit anderen G-Protein-gekoppelten Rezeptoren (GPCR) interagiert. Die Analyse von Coimmunpräzipitaten deutet auf eine Homodimerisierung der beiden CXCR3 Splicevarianten CXCR3A und CXCR3B, sowie auf die Heterodimerbildung von CXCR3A und CXCR3B mit sich, sowie jeweils mit CCR2, CCR3, CCR5 und den Opioidrezeptoren MOR und KOR hin. Die getestete Methode des Fluoreszenz-Resonanz-Energietransfers (FRET) erwies sich jedoch als ungeeignet zur Untersuchung von CXCR3, da dieser in HEK293T-Zellen nicht korrekt transient exprimiert wurde. Insgesamt deuten die Ergebnisse dieser Arbeit darauf hin, dass das CXCR3-Chemokinsystem eine zentrale Rolle in unterschiedlichen, die Neointimabildung beeinflussenden Prozessen spielt. Damit könnten der CXCR3 und insbesondere das Chemokin CXCL10 interessante Zielmoleküle in der Entwicklung neuer verbesserter Therapien zur Verhinderung der Restenose darstellen.<br>Restenosis represents a central problem after coronary angioplasty procedures and is caused by intimal hyperplasia, also called neointima, as a result of transdifferentiation, proliferation of vascular smooth muscle cells and secretion of extracellular matrix. Formation of reactive oxygen species (ROS) and inflammation after vascular injury caused by angioplasty are discussed as early inducers of neointima formation. In several projects the processes causing the development of intimal hyperplasia were investigated. First of all, the impact of inflammation and ROS in neointima formation was investigated using the mouse femoral injury model. The mitochondrial superoxide dismutase mimetic mitoTEMPO could reduce neointima formation better than the global ROS scavenger N-acetylcystein. However, the strongest reduction of neointima formation was achieved by the treatment with the immunosuppressant rapamycin. Interferon-γ(INFγ) is a major cytokine of the Th1 immune response. It is released as a result of vessel injury and induces the proinflammatory chemokines CXCL9 (MIG, Monokine Induced by INF), CXCL10 (IP-10, INF inducible Protein of 10 kDa) and CXCL11 (I-TAC, Interferon inducible T-cell-Chemoattractant), which are ligands of the CXC chemokine receptor 3 (CXCR3) and by this chemotactically recruit CXCR3 positive cells to the site of vessel injury. In this work the special role of CXCL10 in restenosis was investigated. Therefore, CXCL10 decient mice underwent the mouse femoral injury model. The vessels were analysed morphometrically and immunohistologically 14 days after injury. CXCL10 deciency lead to decreased neointima formation that correlated with a reduced recruitment of inflammatory cells as well as diminished numbers of apoptotic and proliferating cells at the site of vessel injury. In addition to inflammation the reconstitution of the endothelium has also impact on the development of restenosis. Interestingly reendothelialisation was strongly improved in CXCL10 decient mice compared to wildtype mice. Obviously the CXCR3 chemokine system is involved in different biological prosesses and impairs neointima formation on one hand by the advancement of inflammation and on the other hand by the suppression of reendothelialisation. In fact the CXCR3 is not only expressed on inflammatory cells but also on endothelial cells. To investigate the role of CXCR3 in inflammation and reendothelialisation separatly the generation of conditional CXCR3 knockout mice with a CXCR3 knockout in T-cells or endothelial cells was started in an additional project. For a better understanding of the molecular mechanisms on which the CXCR3 mediates its biological functions the protein-protein interactions of the CXCR3 with other G-protein coupled recteptors (GPCR) was analysed. Coimmunoprecipitation showed homodimerization of the CXCR3 splice variants CXCR3A and CXCR3B, as well as heterodimerization of CXCR3A and CXCR3B with each other and with the chemokine receptors CXCR4, CCR2, CCR3, CCR5 and the opioid receptors MOR and KOR. The additional tested Fluorecence resonance energy transfer (FRET) method proved to be not suitable to measure interactions of CXCR3, since this receptor could not be expressed correctly on the cell surface after transient transfection. To summarise, the results indicate that the CXCR3 chemokine system plays a central role in different processes that mediate neointima formation. Thus, the CXCR3 and especially the chemokine CXCL10 could be interesting therapeutic targets in the development of new or improved treatments to reduce the risk of restenosis.

H2S is a biologically relevant signaling gas that is endogenously produced throughout the body. The (patho)physiological roles of H2S have led researchers to develop various compounds that decompose to release H2S (H2S donors) for exogenous H2S administration. However, many small molecule H2S donors suffer from poor solubility, low stability, and lack of control over H2S release rates. As a result, there has been an increasing interest in utilizing supramolecular materials for exogenous H2S delivery. With growing potential applications of supramolecular H2S-releasing materials, it is important to explore their properties, e.g., solubility and stability under physiological conditions. We investigated the hydrolytic stability over a range of pH conditions of a series of peptides containing H2S-releasing S-aroylthiooximes (SATOs). The SATO-peptides showed structure–reactivity relationships with SATO ring substituents playing a crucial role in hydrolysis rates. Electron-donating substituents accelerate the rate of hydrolysis while electron-withdrawing substituents slows it down. We also explored their hydrolysis mechanisms at different pH values. SATO-peptides were then used to form hydrogels at 1 wt.% triggered by Ca2+. Hydrogels can be applied directly at a site of interest, potentially improving the efficacy of H2S compared with small molecule donors that diffuse away. We developed a H2S-releasing hydrogel capable of slowly releasing H2S locally to test its efficacy on intimal hyperplasia. The hydrogel delivered H2S over the period of several hours and inhibited the proliferation of human vascular smooth muscle cells (VSMCs) significantly better than fast-releasing NaSH salts. This study shows a promising application of supramolecular H2S-releasing materials over widely used sulfide salts. The macroscopic properties of peptide hydrogels could be further modulated to achieve additional control over the H2S release properties. We synthesized a series of peptide hydrogels incorporating different linker segments to study their effects on hydrogelation properties. Most peptides formed hydrogels but with significantly different rheological behavior. We found that peptides with flexible linkers such as ethyl, substituted O-methylene, and others, formed stronger hydrogels compared to those with more rigid linkers. Interestingly, we found that stiffer hydrogels released H2S over longer periods than softer ones by retarding the diffusion of a thiol trigger, likely due to bulk degradation of the soft gels but surface erosion of the stiff gels as they release H2S.<br>Doctor of Philosophy<br>H2S has long been known as a foul smelling gas until it was discovered that it is endogenously produced throughout the body and plays many (patho)physiological roles. Therapeutic benefits of H2S have led researchers to develop various compounds that release H2S (H2S donors) for exogenous H2S administration. However, many small molecule H2S donors suffer from poor solubility, low stability, and unregulated H2S release. As a result, there has been an increasing interest in utilizing materials for exogenous H2S delivery. With growing potential applications of H2S-releasing materials, it is important to explore their properties, e.g., solubility and stability under physiological conditions. We investigated the stability of a series of peptides containing H2S-releasing S-aroylthiooximes (SATOs) over a range of pH conditions. The stability of SATO-peptides was dependent on chemical makeup of the SATO part of the peptides. We also explored their hydrolysis mechanisms at different pH values. SATO-peptides were then used to form hydrogels triggered by Ca2+. Hydrogels can be applied directly at a site of interest, potentially improving the efficacy of H2S compared with small molecule donors that diffuse away. We developed a H2S-releasing hydrogel capable of slowly releasing H2S locally to test its efficacy on intimal hyperplasia. The hydrogel delivered H2S over the period of several hours and inhibited the proliferation of human vascular smooth muscle cells (VSMCs) significantly better than fast-releasing NaSH salts. This study shows a promising application of supramolecular H2S-releasing materials over widely used sulfide salts. The macroscopic properties of peptide hydrogels could be further modulated to achieve additional control over the H2S release properties. We synthesized a series of peptide hydrogels incorporating different linker segments to study their effects on hydrogelation properties. Most peptides formed weak to strong hydrogels with calcium chloride.We found that peptides with flexible linkers formed stronger hydrogels compared to those with more rigid linkers. Interestingly, we found that stiffer hydrogels released H2S over longer periods than softer ones.

OBJETIVO: analisar, por meio de morfometria digital, a reação intimal presente na artéria carótida de suínos submetidos à angioplastia isoladamente e à angioplastia seguida de implante de stent de cromo - cobalto. MATERIAIS E MÉTODOS: em oito suínos sadios foi realizada a angioplastia isolada da artéria carótida comum (ACC) direita e angioplastia com implante de um stent de cromo – cobalto expansível por balão na artéria carótida comum esquerda. Após período de quatro semanas, os animais foram submetidos à eutanásia para a retirada de amostras de tecido arterial e preparo de lâminas histológicas divididas do seguinte modo: grupo 1, segmento médio da artéria carótida comum direita (angioplastia isolada); grupo 2, segmento médio da artéria carótida comum esquerda em localização “intra stent”. As imagens das lâminas foram digitalizadas e analisadas por programa de morfometria digital com cálculo da área luminal, área da camada íntima e área da camada média dos cortes histológicos. A análise estatística foi realizada através da média e desvio padrão das áreas em cada grupo, utilizando-se Teste t de Student. O valor de p<0,05 foi considerado significativo. RESULTADOS: na análise das médias das áreas obtidas, foi encontrada maior hiperplasia em resposta ao implante de stent e diferença estatisticamente significativa quando realizada a comparação entre a área do lúmen (5,841 x 106μm2 X 1,287 x 106μm2), da lâmina elástica interna (6,566 x 106μm2 X 1,287 x 106μm2) e lâmina elástica externa (9,832 x 106μm2 X 4,559 x 106μm2) dos dois grupos (ATP + STENT X ATP; medidas em micrômetros quadrados). Não se observou diferença significativa do ponto de vista estatístico quando se realizou a comparação entre as camadas médias dos dois grupos (3,266 x 106μm2 X 3,271 x 106μm2). CONCLUSÃO: o implante de stent de cromo-cobalto expansível por balão na ACC do suíno gerou um espessamento intimal maior do que aquele produzido apenas pela angioplastia, porém este não foi suficiente para afetar o lúmen arterial.<br>OBJECTIVE: to analyze, through digital morphometry, the intimal reaction in the carotid artery of pigs submitted to isolated angioplasty and angioplasty followed by implantation of cobalt-chromium stent. MATERIALS AND METHODS: eight healthy pigs had their common carotid artery (CCA) submitted to isolated angioplasty in the right side and angioplasty plus stenting in the left side. Four weeks latter, all animals were submitted to euthanasia for arterial tissue sampling and preparation of histological blades sorted as follows: group 1, middle segment of common right carotid artery (isolated angioplasty); group 2, middle segment of common left carotid artery (intra-stent). Blade images were scanned and analyzed through a digital morphometry program with calculation of luminal, intimal and media layers area in the histological sections. The statistical analysis was performed through mean values and standard deviations of the areas in each group, using the Student’s t-Test. The value of p<0.05 was considered significant. RESULTS: When compare to angioplasty alone, the stent group showed greater hyperplasia in response to implantation regarding the lumen area (5.841 x 106μm2 X 1.287 x 106μm2), the internal elastic lamina area (6.566 x 106μm2 X 1.287 x 106μm2) and the external elastic lamina area (9.832 x 106μm2 X 4.559 x 106μm2). No statistically significant difference was observed when comparing the media layers of both groups (3.266 x 106μm2 X 3.271 x 106μm2). CONCLUSION: angioplasty followed by the implantation of a cobalt-chromium balloon expandable stent in the CCA of the pig creates more intimal thickening than angioplasty alone. Nevertheless intimal thickening was not enough to affect the luminal area thanks to a positive elastic remodeling effect.

Background Morbidity and mortality from Thoraco-abdominal aneurysms are tremendous. Preoperative assessment in predicting cardiac and pulmonary risk factors in order to reduce cardiopulmonary complications, paraplegia, and renal failure are the main determinants of postoperative mortality and therefore gained substantial attention during the last decades.(Jacobs MJ et al.,2007) Left heart by-pass, CSF drainage and epidural cooling have significantly reduced the paraplegia rates. Monitoring MEPs allowed detection of cord ischemia,(spinal cord ischemic injury, SCII) guiding aggressive surgical strategies to restore spinal cord blood supply and reduce neurological deficit. It’s believed that these protective measures should be included in the surgical protocol of TAAA repair type II cases. (Jacobs MJ et al.,2007) Renal and visceral ischemia can be reduced significantly by continuous perfusion during TAAA repair. (Jacobs MJ et al.,2007) Obviously endovascular modalities have been successfully applied in TAAA patients, the majority apart of hybrid procedures. Technological innovations will eventually cause a shift from open to minimal invasive surgical repair. (Jacobs MJ et al.,2007) The multilayer aneurysm repair system (MARS) is a flow modulator and part of the fluid smart© technology platform developed by Cardiatis, Isne-Belgium. The MARS is self-expanding and composed of multiple cobalt alloy interconnected braided layers the 3D geometrical configuration gurantees an optimal porosity range for stent from 2 to 50mm in diameter providing unique flow modulating features. The innovative multilayer flow modulating device offers a paradigm shift approach to the treatment of these complicated aneurysms by physiologically (rather than mechanically ) excluding aneurysms from the circulation, while keeping branches patent and preserving critical collateral circulation.(C.Vaislic et al.,2011) Its 3D geometrical and structural gives rise to several important hemodynamic and biological effects i.e, in a secular aneurysm it reduces the vortex velocity within the aneurysm sac creating a remodeled organized thrombus, it transforms turbulent flow into laminar flow preserving collateral circulation when over stenting collaterals or in a fusiform aneurysm, it accelerates and channels the flow into a branching aneurysm open branch enhancement and accelerates shear flow along the parent vessel, resulting in inhibition of intimal hyperplasia. (C.Vaislic et al.,2011) Theoretical basic principles of the device are very attractive and the most important of which is preserving the collaterals and improving their flow. Sac thrombosis and sac shrinkage don’t  usually occur immediately, several factors could play a role as collateral branches and this should be studied and determined preoperatively.(M.Henry,2011) Preliminary clinical results are satisfactory and promising but larger experience and longer follow up are still needed. (M.Henry,2011) Hypothesis The results of endovascular treatment for thoraco-abdominal aneurysms are now comparable with the open surgical procedure. However, endovascular repair in the thoraco-abdominal aorta has been limited due to the complexity of keeping the side branches (intercostals ,renal, visceral) perfused. Attempts are being made to adapt endoluminal stent-graft by using custom fenestrations and branched graft. While preliminary data showed concept feasibility, this technique is still investigational and requires highly skilled operator and custom made devices for each patient. Aim of the work 1- To study the availability of the new technique in management of TAAA. 2- To shed lights on the technique of deploying the new device. 3- To review the outcome and complications of this new device before introducing this technique as a standard utility. 4- To shed lights over the new evolving biomedical technology in vascular surgery This will be supported by the French STRATO trial and by a number of cases done at multi- european centers Keywords Thoraco-abdominal aneurysms, Multilayer Aneurysm Repair System (MARS) Intimal hyperplasia, Sac shrinkage, Sac thrombosis, Ischemia References 1- Jacobs MJ, Mommertz G, koeppel TA, Langer S, Nijenhuis RJ, Mess WH, Schurink GW. Surgical repair of TAAA. J Cardiovasc Surg Torino(2007) Feb; 48 (1) :49-58 2- C.Vaislic, A.Benjelloun ,J.-N.Fabiani, J.-F.Bonneville, S.Chocron. multilayered flow modulator treatment of thoraco-abdominal aortic aneurysms.Controversies and vascular updates (2011)JAN;74:443-449 3- M.Henry,MD. The Multilayer Stent. First human study.(2011), ISET presentation.

L’athérosclérose et ses complications demeurent une des causes majeures de décès dans les pays industrialisés. L’angioplastie coronarienne n’a pas cessé de progresser et atteint de hauts niveaux de réussite technique. Toutefois, des obstacles majeurs au succès à long terme de l'angioplastie persistent (Davies et Hagen, 1994). C’est le cas de la resténose. Plus de 30 à 60% des artères coronariennes ayant subies une angioplastie sont des cas de resténose, survenant durant la première année de la procédure (Kitazume et al, 1995). Plusieurs facteurs de risques menant vers la resténose post-angioplastie ont été relevés (Lau et Sigwart, 1993). L’hyperplasie intimale et le remodelage artériel sont identifiés comme les deux causes responsables de ce processus multifactoriel (Geary et al., 1998). Hypothèse de recherche : Des évidences suggèrent une implication de divers médiateurs vasoactifs et facteurs de croissances dans le processus resténotique. L’angiotensine II et les endothélines se présentent comme des éléments clés dans la prévention et l'atténuation de l'hyperplasie intimale dans les modèles expérimentaux. Objectif : Investiguer les rôles de ces deux familles de médiateurs dans l’hyperplasie intimale afin de mieux comprendre leurs mécanismes d’actions lors de la resténose post-angioplastie. Buts: 1) Vérifier les effets des bloqueurs de l'angiotensine II (antagonistes et inhibiteurs de l'enzyme de conversion de l’angiotensine II) dans le modèle de la carotide de rat; 2) Vérifier les effets des bloqueurs des endothélines (antagonistes et inhibiteurs de l'enzyme de conversion de l'endothéline) dans le modèle de la carotide de rat; 3) Tester les approches ayant montré une efficacité significative dans un nouveau modèle animal d’angioplastie: le modèle de la carotide de furet; 4) Développer de nouveaux outils et de nouvelles approches pharmacologiques (nouveaux composes, combinaisons, et formulations de microsphères); 5) Mettre au point un modèle intégré d’analyse pharmacotoxicologique chronique de moyenne durée chez le rat. Résultats: Les résultats révèlent un rôle des antagonistes du récepteur AT[indice inférieur 1], mais non ceux du récepteur AT[indice inférieur 2], de l'angiotensine II contre l'hyperplasie intimale dans le modèle d’angioplastie par ballonnet chez le rat mais pas chez le furet. L’inhibition limitée à 50% n’est pas améliorée, à dose égale, par une approche locale que procure l’utilisation des microsphères. Quant aux bloqueurs du système des endothélines, ils démontrent une efficacité similaire à atténuer l’hyperplasie intimale à celles des antagonistes de l’angiotensine II, dans le même modèle du rat. Le développement de la méthode d’administration continue a, de plus, permis de valider le prélèvement sanguin et les mesures hémodynamiques de façon chronique. Conclusions : Au moins deux systèmes sont impliqués dans l’hyperplasie intimale chez le rat. L’angiotensine II ne semble pas avoir un rôle dans l’hyperplasie intimale du furet même si ce modèle démontre une hyperplasie intimale bien évidente suite à la dilatation artérielle, telle qu’observée par d’autres équipes chez le porc. Le rat demeure un modèle utile mais limitée quant à l'interprétation et l'extrapolation que l'on peut en faire vers une application clinique, puisqu’une même approche thérapeutique est inefficace chez le furet. Une formulation permettant une action localisée, telle que l'application de microsphères contenant le médicament, n’a pas démontré une efficacité supérieure. Les effets anti-prolifératifs de bloqueurs des endothélines, dissociables de ceux relies aux bloqueurs de I’angiotensine II, suggèrent qu’une approche combinée pourrait mener à une efficacité supérieure contre l’hyperplasie intimale.

Die subendotheliale myointimale Hyperplasie (MIH) stellt eine der Hauptursachen für die Ausbildung von Stenosen und Verschlüssen im Bereich von Anastomosen dar. Besondere Bedeutung hat die MIH in termino-lateralen Anastomosen. An der Entstehung der MIH sind hämodynamische Faktoren entscheidend beteiligt. Ausgehend von der Annahme, daß eine Veränderung der Anastomosenform die Hämodynamik beeinflussen kann, wurde in der vorliegenden Arbeit eine neue Anastomosenform, die femorocrurale Patchprothese (FCPP), entwickelt mit dem Ziel einer Optimierung der Strömungsverhältnisse im Anastomosenbereich. In einem hydrodynamischen Kreislaufmodell wurden elastische, transparente Silikonmodelle von termino-lateralen Anastomosen mit einem blutanalogen Newtonschen Fluid (Glycerol-Wasser-Gemisch) unter Simulation der femorocruralen Druckkurve pulsatil bei Variation der Strömungsbedingungen perfundiert. Die konventionellen und klinisch erprobten Anastomosenformen (termino-laterale Anastomose, Composite Bypass, Linton Patch, Miller Collar, Taylor Patch) wurden in vitro mit der FCPP-Anastomose und zwei Modifikation dieser Anastomosenform verglichen. Die Visualisierung des Strömungsfeldes wurde mit drei verschiedenen Methoden erreicht. Bei der farboptischen Methode wurde die Verteilung und Bewegung von Farbteilchen im Anastomosenbereich nach Injektion in das strömende Fluid mittels Video aufgezeichnet. Für die Ultraschalluntersuchung mittels hochauflösender farbcodierter Dopplersonographie (FKDS) wurde das Fluid mit Sephadex-Partikeln dotiert. Sämtliche Ultraschalluntersuchungen wurden ebenfalls mittels Video aufgezeichnet. Die Dopplerspektren korrespondierender Punkte in den Randzonen der Anastomosen wurden off-line der Fast Fourier Analyse (FFT) unterzogen und diese dreidimensional dargestellt. Die semiquantitative Analyse anhand der farboptischen Methode zeigte eine signifikante Verringerung bis Elimination MIH-assoziierter Strömungsphänomene in der FCPP-Anastomose und deren Modifikationen. Die Ausprägung der einzelnen Strömungsphänomene war abhängig von der Reynolds-Zahl und von dem Verhältnis von proximalem und distalem Stromzeitvolumen. Als Vorteil der FCPP erwies sich die Möglichkeit der Anpassung ihrer Form an die entsprechende Flußsituation durch Modifikation der Gabelform mit annähernd laminarer Strömung im gesamten Anastomosenbereich. Die FKDS bestätigte die Ergebnisse der farboptischen Methode. In der Peak-Systole und am Beginn der Diastole waren Rezirkulationszonen und Zonen niedriger Strömungsgeschwindigkeit in der FCPP am geringsten ausgeprägt oder nicht mehr nachweisbar (modifizierte FCPP). Der Vergleich der 3D-Darstellungen der FFT der Dopplerspektren zeigte niedrige Frequenzen und damit Flußgeschwindigkeiten und partiell eine Strömungsumkehr nahezu konstant in allen Anastomosenformen außer der FCPP und deren Modifikationen. In einer prospektiven Studie zum klinischen Einsatz der FCPP Anastomose wurden im Zeitraum von 6 / 1992 bis 7 / 1998 135 PTFE-Prothesenbypass-Rekonstruktionen mit distaler FCPP Anastomose bei 129 Patienten im klinischen Stadium III und IV der paVK analysiert. Die kumulativen primären und sekundären 1-, 2-, 3-, 4-, und 5-Jahres-Offenheitsraten nach Kaplan / Meier betrugen jeweils 63,0%, 44,9%, 35,7%, 33,1% und 27,6% bzw. 74,5%, 55,2%, 44,8%, 43,0% und 37,6%. Die kumulativen 1-, 3-, und 5-Jahres-Wahrscheinlichkeiten für den Erhalt der Extremität betrugen jeweils 86,8%, 79,2% und 77,5%. Eine Optimierung des Strömungsverhaltens innerhalb der Anastomosenregion ist in der FCPP derart möglich, daß der Anteil der für die Pathogenese der MIH ursächlichen Strömungsmuster minimiert wird. Diese Anastomose ist klinisch anwendbar und führt bei ausschließlicher Verwendung von ePTFE als Bypassmaterial im femorodistalen Bereich zu akzeptablen Langzeitergebnissen, die denen bei Anwendung von alternativ möglichen Venenpatchplastiken nicht nur vergleichbar, sondern teilweise überlegen sind.<br>The subendothelial myointimal hyperplasia (MIH) represents one of the main etiological factors in the formation of stenoses and occlusions of vascular anastomoses. MIH plays a role especially in termino-lateral anastomoses. Hemodynamic factors have a decisive impact on the development of MIH. Assuming that changes in the morphology of the anastomoses influence the hemodynamics, a novel anastomosis form, the femorocrural patch prosthesis (FCPP), was developed with the goal of optimizing the blood flow-dynamics within the anastomotic site. In a hydrodynamic circulation model, various elastic, transparent silicon phantoms of termino-lateral anastomoses were perfused with a Newton fluid blood analog (glycerol-water mixture) while simulating the femorocrural pressure curve in a pulsatile manner under variation of the flow conditions. The conventional and clinically tested anastomosis forms (termino-lateral anastomosis, composite bypass, Linton patch, Millar collar, Taylor patch) were compared with the FCPP-anastomosis and two modifications of the FCPP in vitro. The visualization of the flow velocity field was achieved using three different methods. By means of the color-optic method, the distribution and motion of color elements in the anastomotic area were video-recorded following injection in the flowing liquid. For the ultrasound examination with high resolution, color-coded Doppler sonography, the fluid was marked with Sephadex particles. All ultrasound examinations were also recorded on video. The corresponding doppler spektrum points in the marginal zones of the anastomosis were subjected to the Fast Fourier Transform (FFT) analysis off-line and then displayed three-dimensionally. The semiquantitative analysis using the color-optic method showed a significant decrease or elimination of MIH-associated current phenomena in the FCPP-anastomosis and its corresponding modifications. The intensity of singular flow phenomena was dependent upon the Reynolds-number and upon the relation of proximal to distal flow volume over time. The possibility to adapt the FCPP to the flow phenomena by modifying the bifurcated form and thereby achieving almost laminar flow in the complete anastomotic area proved to be advantageous. The color-coded Doppler sonography confirmed the results of the color-optic method. During peak systole and at the beginning of the diastole, recirculation zones and zones with low flow velocity were least intense in the FCPP or not detectable at all (in the modified FCPPs). The comparison between the FFT 3D-reconstructions from the Doppler spektrum showed low frequencies and thus flow velocities and a partial flow reversal almost constantly in all anastomosis forms except the FCPP and its modifications. In a prospective study on the clinical application of the FCPP anastomosis during the period of June 1992 to July 1998, 135 ePTFE prosthetic bypass reconstructions with distal FCPP anastomosis were analyzed in 129 patients suffering from peripheral arterial occlusive disease stages III and IV. The cumulative primary and secondary 1-, 2-, 3-, 4-, and 5-year patency rates calculated with the Kaplan / Meier method were 63.0%, 44.9%, 35.7%, 33.1%, and 27.6%, and 75.5%, 55.2%, 44.8%, 43.0% and 37.6%, respectively. The cumulative 1-, 3-, and 5-year probabilities for limb salvage were 86.8%, 79.2% and 77.5%, respectively. As a result of anastomotic engineering, FCPP optimizes the flow properties within the anastomosis region to such an extent that the pathogenetic role of flow disturbances is minimized. This anastomosis is clinically employable when using ePTFE as bypass material in femoro-distal vascular reconstruction and leads to acceptable long-term results that are not only comparable but sometimes superior to the results obtained when using alternative vein cuff techniques.

L'angioplastie avec pose de stent est le traitement de prédilection de la plaque d'athérome symptomatique. Une complication fréquente de cette intervention est la resténose intra-stent qui mène à une nouvelle obstruction de la lumière artérielle. Cette pathologie repose sur le processus d'hyperplasie intimale, combinaison d'une inflammation locale et d'une modulation phénotypique des cellules musculaires lisses (CML) de la paroi artérielle. Afin de pallier à cette pathologie, les stents ont été recouverts d'agents anti-prolifératifs. Bien qu'efficace dans la prévention de la resténose, l'utilisation de ce type de stents a été associée à une augmentation de la fréquence de thromboses à long terme, probablement en lien avec un défaut de la cicatrisation de l'endothélium. Il est donc impératif de trouver des alternatives thérapeutiques pour la prévention de la resténose intra-stent. Dans ce contexte, ce travail de thèse s'intéresse à l'isoforme gamma de la famille des phosphoinositide 3-kinases (PI3K gamma). La PI3K gamma est très exprimée dans le compartiment hématopoïétique, ce qui lui confère des rôles dans diverses pathologies inflammatoires. Elle est également présente à un plus faible niveau dans le système cardiovasculaire, dont les CML artérielles. D'abord, ce travail a contribué à montrer in vitro que la PI3K gamma, surtout connue pour ses fonctions immunes, participait à la migration des CML artérielles en aval de la chimiokine MCP-1, et indirectement en réponse au PDGF. Antérieurement, la PI3K gamma avait été identifiée au laboratoire comme un acteur déterminant dans les processus immuns de l'athérosclérose, pathologie inflammatoire de la paroi artérielle. Au vu de l'implication cruciale de MCP-1 et du PDGF, de la migration des CML, mais aussi de l'inflammation dans l'hyperplasie intimale, nous avons donc proposé la PI3K gamma comme un acteur potentiel dans cette pathologie in vivo. Ensuite, un modèle d'hyperplasie intimale murin a été adapté au laboratoire afin d'évaluer l'implication de la PI3K gamma. Elle a effectivement pu être incriminée dans ce processus via son activité inflammatoire, grâce à deux modèles d'invalidation génétique et une stratégie d'inhibition pharmacologique de la kinase. Notre travail a également contribué à la compréhension générale des mécanismes de la pathologie : nous avons en effet identifié un rôle délétère des lymphocytes T CD4+ associé à une augmentation drastique de la production de cytokines de type Th1 et Th17 dans l'artère lésée. Dans ce contexte, la PI3Kgamma est capable de moduler la réponse de type Th1. Enfin, au vu des effets secondaires des traitements actuels sur la cicatrisation endothéliale, l'impact d'une invalidation de la PI3K gamma dans ce processus a été étudié. L'absence d'activité PI3K gamma dans le compartiment hématopoïétique a permis d'accélérer la cicatrisation endothéliale. Dans ce même contexte, l'effet d'un revêtement par de nouveaux biomatériaux sur le comportement des cellules endothéliales in vitro a également été évalué, apportant des résultats prometteurs. L'ensemble de ces données désigne la PI3K gamma comme une cible thérapeutique intéressante pour la prévention de la resténose intra-stent puisque son invalidation atténue l'hyperplasie intimale tout en accélérant la cicatrisation endothéliale après une lésion artérielle<br>Angioplasty with stent placement is the most used treatment against symptomatic atherosclerosis. A frequent side effect of this intervention is in-stent restenosis, which leads to a de novo obstruction of the arterial lumen. This pathology is characterized by intimal hyperplasia, a combination of a local inflammation and a phenotypic modulation of medial smooth muscle cells (SMC). In order to overcome this complication, stents have been coated with antiproliferative drugs. Although these devices proved to be efficient in retenosis prevention, their use has been statistically associated with an increase in long term thromboses, probably linked to a defect in endothelial healing. So it is necessary to find alternative treatments for in-stent restenosis prevention. In this context, this work takes an interest in the gamma isoform of the phosphoinositide 3-kinase (PI3K) family. PI3K gamma is highly expressed in the hematopoietic compartment and displays a large variety of functions in numerous inflammatory pathologies. This protein is also expressed at a lower level in the cardiovascular system, in particular in arterial SMC. Firstly, the present work has contributed to show that PI3K gamma, well-known for its immune functions, participates to arterial SMC migration in response to the chemokine MCP-1, and indirectly downstream PDGF. Previously, PI3K gamma has been identified in the laboratory as a key player in the immune processes governing atherosclerosis, a chronic inflammatory disease of the arterial wall. Given the crucial involvement of MCP-1 and PDGF, SMC migration and inflammation in intimal hyperplasia, we proposed PI3Kgamma as a potential candidate in the development of this process in vivo. Secondly, a mouse model of intimal hyperplasia has been developed in the laboratory to evaluate the involvement of PI3K gamma. Indeed, we were able to prove that PI3K gamma participated to inflammatory mechanisms of intimal hyperplasia. Our work contributed to the global understanding of the mechanisms underlying this pathology : we have identified a deleterious role of CD4+ T cells accompanied by a drastic increase in Th1 and Th17 cytokines in response to arterial injury. In this context, PI3K gamma was able to modulate Th1 response. Finally, given the side effects of current restenosis treatments upon endothelial healing, the impact of PI3K gamma invalidation in this process has been investigated. Absence of immune PI3K gamma activity enabled an acceleration of endothelial healing. For the same purpose, endothelial cells behavior on new hydrogenocarbonated materials has been evaluated in vitro, bringing promising results. Altogether, these data pinpoint PI3Kgamma as an interesting therapeutic target for in-stent restenosis prevention since its invalidation is both able to prevent restenosis and to accelerate endothelial healing after arterial injury

Introdução: Apesar dos avanços significativos no tratamento endovascular das doenças arteriais coronarianas e periféricas, a reestenose intra-stent continua sendo o principal limitante a médio prazo desses procedimentos. O mecanismo da reestenose intra-stent é principalmente a hiperplasia intimal, já que o stent impede a retração elástica aguda e resiste ao remodelamento geométrico negativo tardio. A hiperplasia intimal ocorre basicamente em resposta à formação de trombo local, à inflamação e às dissecções intimais e mediais secundárias à injúria causada pelo stent, sendo o grau de resposta intimal a base dos efeitos a longo prazo. O uso de stents com hastes menores e revestidos com drogas ou polímeros tem sido considerado uma nova alternativa para a prevenção da reestenose intra-stent. Objetivo: Analisar a resposta arterial ao implante de stent de cromo-cobalto sem e com revestimento de polímero Camouflage® em artérias carótidas de suínos, utilizando os seguintes parâmetros histológicos: grau de endotelização, conteúdo de células musculares lisas, grau de angiogênese, conteúdo de fibrina, grau de inflamação e injúria; além da análise histomorfométrica. Método: Stents balões-expansíveis de cromo-cobalto ( 8 stents CC Flex e 5 stents CC Flex Proactive) de 4 x 16 mm foram implantados em artérias carótidas comuns de oito suínos jovens, sendo um stent liberado em cada artéria. Após 30 dias, as artérias contendo os stents foram removidas, fixadas e coradas pelos métodos de hematoxilina/eosina e Verhoeff/Van Giesson. O segmento arterial contendo o stent foi dividido em 3 blocos distintos: proximal, médio e distal. Os cortes histológicos foram obtidos utilizando-se micrótomo de impacto (Polycut S, Leica, Alemanha) equipado com navalha de tungstênio de 16 cm, tipo D (Leica, Alemanha), com 5 ^m de espessura. A navalha de tungstênio mantém as hastes dos stents intactas nas secções transversas, minimizando os artefatos potenciais causados pela retirada dos stents. A avaliação foi realizada através de critérios histológicos e histomorfométricos. Resultados: Todos os stents foram implantados com sucesso e sem dificuldades técnicas. A análise histológica em 30 dias evidenciou alto grau de endotelização em todos os segmentos avaliados e leve à moderada infiltração de células musculares na íntima. Observou-se baixo grau de angiogênese em cerca de 50% dos segmentos avaliados e ausência completa de deposição de fibrina em pelo menos 80%, com distribuição semelhante entre os grupos. A resposta inflamatória e o grau de injúria causadas pelas hastes dos stents também foram discretas e similares entre os grupos e não houve correlação entre resposta inflamatória e injúria e desses parâmetros com a área de neoíntima. O grau de obstrução neo-intimal identificada neste período foi pequeno (15,1% +/- 8,38 CC Flex x 15,5%+/- 5,39 CC Flex ProActive) e estatisticamente não significativo entre os grupos (p=0,785). Conclusão: Os achados deste estudo experimental sugerem que o uso de stents de cromo-cobalto revestidos com polímero Camouflage® em artérias carótidas de suínos parece estar associado, pelo menos no curto prazo, a uma resposta histológica semelhante àquela encontrada após o implante de stents de cromo-cobalto não revestidos. Neste período não se observou uma menor hiperplasia intimal em virtude do revestimento de polímero.<br>Introduction: Despite all the advances in the endovascular treatment of coronary and peripheral artery diseases, in-stent restenosis is still the main limiting factor of these procedures in the medium and long-term. The mechanism of in-stent restenosis is mainly the intimal hyperplasia, as the stent prevents acute elastic recoil and later negative geometric arterial remodeling. Intimal hyperplasia occurs basically in response to the formation of local thrombus, inflammation and intimal and medial dissections secondary to the injury caused by the stent, with the degree of intimal response being the cause of long-term effects. Coating drug-eluting stents with polymers and drugs with thinner struts have been considered a new alternative for in-stent restenosis prevention. Objective: Analyse the arterial response to the cobalt-chromium stent implant with and without polymer coating Camouflage® in carotid arteries of pigs, using the following histological parameters: degree of endothelialization, smooth muscle cells (SMC) content, degree of angiogenesis, intimal fibrin content, degree of inflammation and injury; plus histomorphometric analysis. Method: Cobaltchromium balloon-expandable stents (8 CC Flex stents and 5 CC Flex Proactive), 4 x 16 mm, were deployed in common carotid arteries of 8 young pigs, with one stent being deployed in each artery. After 30 days, the arteries containing the stents were removed and underwent fixation and staining using the hematoxilin/eosin and Verhoeff /Van Giesson methods. The arterial segment containing the stent was divided into 3 distinct portions: proximal, middle and distal. The histological sections were obtained using impact microtome (Polycut S, Leica, Germany), equipped with a 16 cm, type D, 5 ^m thick tungsten knife (Leica, Germany). The tungsten knife maintains the stent shaft intact in cross sections, minimizing the potential artifacts caused by stent removal. The evaluation was carried out using histological and histomorfometric criteria. Results: All the stents were deployed with success and with no technical difficulties. The histological analysis performed after 30 days showed a high level of endothelialization in all the evaluated portions and mild to moderate infiltration of the SMC in the intima layer. A low level of angiogenesis of about 50% of the evaluated portions was observed and a complete absence of fibrin deposition in at least 80% of the portions, with similar distribution among the groups. The inflammatory response and the level of injury caused by the struts of the stents were also minimum and this was similar among the groups. There was no correlation between inflammatory response and injury and between the two latter parameters and the neo-intima area. The level of neo-intimal obstruction identified in this period was small (15,1% +/- 8,38 CC Flex x 15,5%+/- 5,39 CC Flex ProActive ) and no statistical significance between the groups (p=0,785). Conclusion: The findings of this experimental study suggest the use of balloonexpandable cobalt-chromium stents coated with polymer Camouflage® in carotid arteries of pigs seems to be associated, at least in the short-term, with a similar histological response to that found in the implantation of non-coated cobalt-chromium stents. In this period, a lower intimal hyperplasia was not observed with polymer coating stents.

Durch die intraoperative Anwendung der extrakorporalen Bypassflow-Messung wurde zum ersten Mal die Messung und Berechnung der hydraulischen Impedanz möglich. Von den in der vorgelegten Untersuchung berücksichtigten Variablen des hydraulischen Impedanzkonzeptes zeigte sich, dass die Phasenverschiebung zwischen Druck- und Flusskurven, gemessen als erster Impedanzphasenwinkel, die größte klinische Bedeutung hatte. Mit Hilfe der Phasenverschiebung wurde die Langzeitprognose von femoro-poplitealen und insbesondere cruralen Bypässen eindeutig vorhersagbar. Sämtliche Bypässe zu Ausstromgebieten, die einen hohen Wellenwiderstand von < -40° aufwiesen, waren innerhalb von 16 Monaten nach der Operation verschlossen. Das Verhalten der Impedanzparameter nach Prostaglandinapplikation bestätigte die bisherigen tierexperimentellen Erfahrungen. Durch die induzierte Vasodilatation kam es neben der Senkung des linearen Strömungswiderstandes auch zu einem deutlichen Abfall des pulsatilen Wellenwiderstandes. Es zeigte sich, dass die isolierte Prostaglandinreaktion ein weiterer Prognosefaktor von cruralen Bypässen war. Sämtliche Rekonstruktionen, die zu Anschlussgefässen geführt wurden, welche keine Prostaglandinreaktion mehr zeigten, waren innerhalb von 9 Monaten postoperativ verschlossen. Hierdurch konnte die Genauigkeit der Vorhersagbarkeit von Bypassverschlüssen noch verbessert werden. Es zeigte sich außerdem in der vorgelegten Studie, dass konventionelle Angiographien weder mit der Prognose der peripheren Rekonstruktionen noch mit den intraoperativ gemessenen linearen oder pulsatilen Widerständen korrelierten. Dieses ist um so mehr von Bedeutung, da die Operationsplanung in der Regel anhand der Angiographien erfolgt. In kritischen Fällen, in denen der periphere Abstrom aufgrund einer schwachen Kontrastierung der Anschlussgefässe nicht sicher zu bestimmen ist, sollten aus diesem Grund zusätzliche dynamische Untersuchungen, wie die farbkodierte Duplexsonographie oder Magnetresonanz-Angiographie erfolgen. Der hohe lineare und pulsatile Widerstand ist zusammen mit der Anastomosengeometrie weiterhin dafür verantwortlich, dass es innerhalb der Anastomosen zu ausgeprägten Flussseparationen kommt. Wie mit Hilfe der Particle Image Velocimetry-Untersuchung gezeigt wurde, finden diese sich innerhalb der klassischen End-zu-Seit-Anastomosen im Bereich der Haube und der Fersenregionen. Darüber hinaus kommt es in Abhängigkeit von der Geometrie zu einer Stagnationszone auf dem Boden der Anastomose. Innerhalb dieser Flussablösungszonen fanden sich Scherstressmuster, die deutlich unterhalb des normalen Wandscherstressniveaus lagen. Zwischen dem Zentralstrom und den Separationszonen ließen sich darüber hinaus Übergangszonen mit deutlich höherem Scherstress nachweisen. Diese Befunde unterstützen die sogenannte low-shear Theorie, welche zur Erklärung der subendothelialen Intimahyperplasie herangezogen wird. Die räumliche Korrelation der typischen Prädilektionsstellen der Intimahyperplasie mit den durch PIV nachgewiesenen Flussablösungen zeigte eine sehr hohe Übereinstimmung. Neben den Hauben- und Fersenseparationen entwickelte sich eine Stagnationszone am Boden des Empfängergefässes. Ein weiterer wichtiger Aspekt in diesem Zusammenhang war der erstmals erhobene Befund, dass in der Übergangszone zwischen Zentralstrom und Separationen normale Wandscherbedingungen vorlagen. Hierbei könnte es sich um das erforderliche Stopsignal für die Endothelzellen handeln, die Stimulation der subendothelialen Myozyten einzustellen.<br>The intraoperative use of the extracorporeal bypass flow method allowed for the first time measurement and calculation of hydraulic impedance. The phase shift between flow and pressure waves, as represented by the first phase angle, showed a strong correlation to graft patency. All grafts presenting with high impedance values of < -40°, were occluded within 16 month after the operation. The reaction of the impedance parameters following application of prostaglandin E1 correlated with the previously known experimental results. The induced vasodilation led to a significant decrease of the first phase angle. The prostaglandin reaction itself also proved to be a valuable prognostic factor. All reconstructions to a vasculature with a negative prostaglandin reaction were occluded within 9 months after the operation. The prostaglandin response therefore provided an enhancement of the prognostic tools associated with graft patency. In addition it was shown, that the conventional angiographies did not correlate with graft prognosis. This is particularly important, since angiographies are used for the preoperative planning of the operation. In critical cases, additional assessment of runoff by means of duplex sonography or magnetic resonance angiography is necessary. The high pulsatile resistance is responsible for flow separation inside distal anastomoses of peripheral bypasses. Using Particle Image Velocimetry it was shown, that significant separation areas were present at the heel, the hood and the floor of the anastomoses. The shear stress inside these areas was significantly lower than normal wall shear stresses. At the transition between separation and mainstream very high shear stresses were present, which could provide the stop signal for the signal cascades, leading to subendothelial hyperplasia, which leads to graft stenoses.

Peripheral vascular grafts are used for the treatment of peripheral arterial disease and arteriovenous grafts for vascular access in end stage renal disease. The development of neo-intimal hyperplasia and thrombosis in the distal anastomosis remains the main reason for occlusion in that region. The local haemodynamics produced by a graft in the host vessel is believed to significantly affect endothelial function. Single spiral flow is a normal feature in medium and large sized vessels and it is induced by the anatomical structure and physiological function of the cardiovascular system. Grafts designed to generate a single spiral flow in the distal anastomosis have been introduced in clinical practice and are known as spiral grafts. In this work, spiral peripheral vascular and arteriovenous grafts were compared with conventional grafts using ultrasound and computational methods to identify their haemodynamic differences. Vascular-graft flow phantoms were developed to house the grafts in different surgical configurations. Mimicking components, with appropriate acoustic properties, were chosen to minimise ultrasound beam refraction and distortion. A dual-beam two-dimensional vector Doppler technique was developed to visualise and quantify vortical structures downstream of each graft outflow in the cross-flow direction. Vorticity mapping and measurements of circulation were acquired based on the vector Doppler data. The flow within the vascular-graft models was simulated with computed tomography based image-guided modelling for further understanding of secondary flow motions and comparison with the experimental results. The computational assessments provided a three-dimensional velocity field in the lumen of the models allowing a range of fluid dynamic parameters to be predicted. Single- or double-spiral flow patterns consisting of a dominant and a smaller vortex were detected in the outflow of the spiral grafts. A double- triple- or tetra-spiral flow pattern was found in the outflow of the conventional graft, depending on model configuration and Reynolds number. These multiple-spiral patterns were associated with increased flow stagnation, separation and instability, which are known to be detrimental for endothelial behaviour. Increased in-plane mixing and wall shear stress, which are considered atheroprotective in normal vessels, were found in the outflow of the spiral devices. The results from the experimental approach were in agreement with those from the computational approach. This study applied ultrasound and computational methods to vascular-graft phantoms in order to characterise the flow field induced by spiral and conventional peripheral vascular and arteriovenous grafts. The results suggest that spiral grafts are associated with advanced local haemodynamics that may protect endothelial function and thereby may prevent their outflow anastomosis from neo-intimal hyperplasia and thrombosis. Consequently this work supports the hypothesis that spiral grafts may decrease outflow stenosis and hence improve patency rates in patients.

碩士<br>國立東華大學<br>生物技術研究所<br>98<br>Caveolin-1 (Cav-1) is identified as the major unit forms scaffold structure, regulates several signaling pathway and abundantly expressed in vascular smooth muscle cell (VSMC). VSMC migration contributes to intimal hyperplasia, which is involved in several vascular diseases including atherosclerosis. Previous study demonstrated that Cav-1-deficient VSMC showed an abnormal increase in proliferation and migration rate as compared with wild-type. The mechanism of Cav-1 in VSMC proliferation is already demonstrated, but the role of Cav-1 in VSMC migration has not been certain. In addition, Pyrogallol, a polyphenol compound, plays an antioxidant character and reduces the risk of cardiovascular disease. This study attempted to dissect the role of Cav-1 in VSMC migration and investigate effect of pyrogallol on VSMC mobility and carotid artery ligation to mimic a neointimal hyperplasia phenotype. The data showed that Cav-1 deficient VSMC down-regulated the mRNA expression of MMP-3, MMP-13 and cox-2 whereas up-regulated of MMP-14 expression. Moreover, the AFM picture also showed that Cav-1 deficient VSMC had less lamellipodia and higher filopodia than wild-type VSMC. Additionally, Pyrogallol could significantly inhibit VSMC migration in the presence and absence of Cav-1 via two different pathways. Pyrogallol inhibited WT VSMC migration by repressing MMP-2 activity and increasing TIMP-1 expression. In contrast, without Cav-1 in VSMC, pyrogallol inhibits its migration via promoting the TIMP-2 expression and down-regulating MMP-1 expression. In vivo study, pyrogallol also could significantly inhibit the intima formation at third week after mouse carotid ligation. At the early stage of carotid ligation in mice, the proMMP-9 was significantly increased from Day 0 to Day 2 and decreased from Day 2 to Day 7 at a time-dependent manner from zymography of serum. Furthermore, MMP-9 activity was elevated from Day 2 to Day 7. However there was no significant difference in both MMP-2 and MMP-9 activity after treated with pyrogallol and doxycycline for 8 times, respectively. The present study suggests that pyrogallol could prevent the severity of neointima hyperplasia via a ROS scavenger in mouse carotid ligation model and has potential anti-atherogenic effects in the treatment of vascular diseases.

Adult cardiovascular disease has its origins in childhood, and adolescence is a critical period in determining lifetime risk. Early changes in arterial structure and function measured non-invasively have prognostic significance. Assessment of vascular structure and function provide an opportunity to test intervention strategies at an age when vascular damage is potentially reversible. Understanding the relative sensitivity of these markers of vascular damage is essential in identifying children at risk and enabling evaluation of clinical and public health interventions. In a cross-sectional study, aortic and carotid intima media thickness were assessed in 66 children with type 1 diabetes and 32 healthy children. Aortic intima media thickness (aIMT) was significantly greater in the children with type 1 diabetes and related to age, glycosolated haemoglobin and low-density lipoprotein cholesterol concentrations. In contrast, there was no significant difference in carotid intima media thickness between groups, suggesting that aIMT is an earlier marker of subclinical atherosclerosis in children with type 1 diabetes. An interventional trial of 22 children with type 1 diabetes was performed to evaluate whether reduction in glucose variability with initiation of continuous subcutaneous insulin infusion (CSII) therapy would improve vascular function. At 3 weeks post commencement of CSII, vascular function improved associated with a reduction in glucose variability; however the effects on vascular function over 6 to 12 months were not sustained, with deterioration of glycaemic control. Finally, in a cross-sectional study, vascular function and structure was assessed in 14 children with congenital adrenal hyperplasia (CAH), a relatively novel patient population, whose risk for atherosclerosis has not been previously investigated. The results from the children with CAH were compared to 28 obese and 53 healthy controls. The children with CAH had evidence of vascular dysfunction, comparable to the obese cohort, despite having a lower body mass index. It was concluded that use of non-invasive ultrasound markers of preclinical atherosclerosis can allow early detection of changes in vascular structure in children at known risk for future atherosclerosis, identify novel groups of children with medical conditions not previously recognized to have future cardiovascular risk, and is a valuable tool that can be used to test interventions in a timely manner.<br>Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2014

(ENGLISH) Background: The investigation of the immunosuppression usage in cardiovascular surgery and interventional cardiology is, at present, concentrated on three main topics: 1) influence on intimal hyperplasia of coronary and peripheral vascular reconstructions 2) influence on rejection of allogeneic vascular grafts and 3) influence on intimal hyperplasia of coronary arteries after endovascular interventions. Modern immunosuppressive drug FK506 (Tacrolimus) could have a positive effect for these indications. In experimental study, FK506 inhibited rejection of arterial allografts and also inhibited intimal hyperplasia in percutaneous coronary interventions. Aims: The purpose of this study was to evaluate the effect of systemic tacrolimus treatment on the process of arterialisation of allogeneic and syngeneic venous grafts in a rat vein-to-artery implantation model. Material and Methods: Lewis (LEW) rats were used as recipients of syngeneic (Lewis) or allogeneic (Brown-Norway; BN) iliolumbar veins which were implanted into abdominal aorta. Recipients were divided into six groups. In groups A, E and F were animals after syngeneic (LEW to LEW) and in groups B, C and D were animals after allogeneic (BN to LEW) transplantations. Animals in the groups C and F had daily intramuscular injections of...

碩士<br>國立臺灣海洋大學<br>生物科技研究所<br>95<br>Abstract In ten major causes of the death in Taiwan, cardiovascular disease and hypertension are all relate to atherosclerosis. The main methods to treat coronary artery disease are percutaneous transluminal coronary angioplasty, with or without stent implantation. But within the following 6 months after the operation, nearly 30 - 50％ of the patients have coronary restenosis. Previously, reports showed that cannabinoid system might play a role in the treatment of atherosclerosis. Delta-9-tetrahydrocannabinol, or 6a, 7, 8, 10a-tetrahydro-6,6,9-trimethyl-3-pentyl-6H-dibenzo[b,d ]-pyran-1-ol, is a substance possessing several stereochemical variants. One of them is (2)-trans-delta-9-tetra-hidrocannabinol, also called dronabinol or D9-THC. Two known cannabinoid receptors are namely CB1 and CB2 receptors. In our study, we investigated the effects of THC on the development of intimal hyperplasia on vessels after angioplasty injury. We used western blot to detect CB1 and CB2 receptors, and observed MAPK signal transduction. Cell proliferation capacity were detected by BrdU incorporation assay. In cell functional assays, we used cell migration assay to observe cell migratory ability in response to THC, CB1 antagonist, and CB2 antagonist. In animal experiments, mice underwent femoral artery wire injury. We used immunofluorescence staining to detect the expression of CB1 and CB2 receptors on injured vessel. Our data demonstrated that in the course of forming atherosclerosis or restenosis, THC probably promoted the proliferation of the vascular smooth muscle cells（SMCs）, and accelerated the progression of restenosis. This effect of THC could be blocked by CB1 and CB2 antagonists, by suppressing SMCs proliferation. Thus, cannabinoids system maybe a valuable target for attenuating the development of intimal hyperplasia, namely restenosis, after vascular injury.

碩士<br>中國醫藥大學<br>中國藥學暨中藥資源學系碩士班<br>100<br>Cerebral and cardiovascular diseases are mainly caused by the pathogenesis of arterial vascular smooth muscle. Paeonia suffruticosa Andr. (abbreviated as PSex)is a well-known traditional Chinese herbal medicine. Ancient Chinese medicine literatures have showed that various effects of this herb on human sickness such as reducing heat, promoting blood flow and eliminating stasis. Paeonol (2′-hydroxy-4′-methoxyacetophenone) is one of the main components extracted from PSex. Paeonol was reported to have multi-biological activites such as anti-inflammatory, anti-allergic, anti-tumor, anti-angiogenic, and anti-oxidative activities. In the present study, the effects and action mechanism of PSex and paeonol on vascular smooth muscle cells proliferation and migration were evaluated. Results from our study revealed that PSex reduced hyperplasia in the mouse carotid ligation model. Low-dose (0.1 g/kg) or high-dose (0.2 g/kg) of PSex decreased the level of intima/media (I/M) ratio in 24 % and 37 % compared with the control group, respectively. PSex reduced PCNA positive cells in a dose-dependent manner and the inhibition rate was 35 % (0.1 g/kg) and 62 % (0.2 g/kg) compared with control. In wound healing assay and transwell migration assay. The results showed that PSex and paeonol extracts significantly inhibited platelet-derived growth factor (PDGF) induced A7r5 cells migration and proliferation. PSex and paeonol decreased Ras, MEK, p-MEK, p-ERK1/2 and MMP-2 and MMP-9protein level in western blotting analysis. In conclusion, results from this study revealed that PSex inhibited 10 carotid-ligation induced intimal hyperplasia; PSex and paeonol both inhibited PDGF –induced A7r5 cell proliferation and migration which might be via the inactivation of MMP2, MMP9, and MEK/ERK pathway

血管內膜的進行性增厚是動脈粥樣硬化的重要標誌，並最終導致閉塞性血管病。血管內膜增生的一個主要因素是血管中膜的平滑肌細胞遷移至內膜層並增殖。大量研究證實，在動脈粥樣硬化的發生發展中，過量產生的活性氧簇（ROS）參與了血管壁的增厚。M型瞬時受體電位通道亞家族的成員TRPM2在血管平滑肌細胞中有表達，它能被ROS激活並對Ca²⁺通透，但其在血管平滑肌中的功能以及與心血管疾病的聯繫尚未見報道。<br>本論文著眼於探討TRPM2在鼠和人血管內膜增生中的作用。用血管外周套管法建立在體齧齒類動脈內膜增生模型。套管放置2周後，大鼠股動脈可見明顯的內膜增厚。免疫染色顯示新生內膜及其鄰近中膜區域內有大量增殖細胞核抗原陽性細胞，提示在增生的動脈中，細胞週期活動增強。動脈內膜和中膜内二氫乙錠螢光信號顯著增強，提示了ROS的過量生成。免疫染色和免疫印跡法均顯示，套管損傷導致TRPM2表達上調。免疫螢光雙標TRPM2與α-平滑肌肌動蛋白顯示內膜區域有大量TRPM2陽性的平滑肌細胞。與正常股動脈中膜平滑肌細胞相比，次黃嘌呤和黃嘌呤氧化酶在套管損傷的動脈來源的新生內膜平滑肌細胞中引起更大幅度的細胞內鈣離子濃度升高，而TRPM2抑制性抗體TM2E3預處理可消除這種差異。套管放置3周可引起小鼠頸動脈新生內膜形成，並伴隨著TRPM2表達上調。敲除TRPM2基因可顯著抑制內膜增生。取冠狀動脈搭橋術後殘餘的大隱靜脈，離體培養2周誘導內膜增生。免疫螢光雙標TRPM2與α-平滑肌肌動蛋白顯示新生內膜內含有大量TRPM2陽性的平滑肌細胞。TM2E3和另一TRPM2抑制劑2-氨乙氧基二苯酯硼酸處理均可有效降低內膜的增生。培養齧齒類主動脈平滑肌細胞，用劃痕試驗和MTT法檢測TRPM2阻斷劑和TRPM2基因敲除對過氧化氫誘導的細胞遷移和增殖的影響。結果顯示，暴露於過氧化氫48小時，細胞的遷移和增殖均明顯加快。TM2E3和2-氨乙氧基二苯酯硼酸處理有效抑制過氧化氫誘導的大鼠主動脈平滑肌細胞遷移和增殖；類似地，TRPM2基因敲除可顯著抑制過氧化氫誘導的小鼠主動脈平滑肌細胞遷移和增殖。<br>以上結果表明，血管內膜增生伴隨著TRPM2表達的上調；TRPM2參與了血管內膜增生以及血管平滑肌細胞的遷移、增殖；抑制TRPM2可能是對抗血管內膜增厚的潛在治療手段。<br>A hallmark in atherosclerosis is progressive intimal thickening, which leads to occlusive vascular diseases. A causation of neointimal hyperplasia is the migration of medial smooth muscle cells (SMCs) to the intima where they proliferate. It is well recognized that excessive production of reactive oxide species (ROS) contributes to vascular wall thickening during arteriosclerotic development. TRPM2, a member of the melastatin-like transient receptor potential channel subfamily, is a Ca²⁺-permeable cation channel activated by ROS and is expressed in vascular smooth muscle cells (VSMCs). The functional properties of TRPM2 in vascular smooth muscle remain to be identified and an association between TRPM2 and cardiovascular diseases has not been reported.<br>In the present study, I investigated the involvement of TRPM2 in rodent and human neointimal hyperplasia. In vivo neointimal hyperplasia in rodent arteries was induced by perivascular cuff placement. After the cuff placement for 2 weeks, rat femoral arteries showed distinct intimal thickening. Immunostaining showed a great number of PCNA-positive proliferating cells in the neointima and its adjacent media region, indicating the enhanced cell cycle activity in the hyperplasic arteries. Dihydroethidium signal was markedly increased in the neointima and media of the cuffed arteries, suggesting that ROS is over-produced. Interestingly, both immunostaining and immunoblot showed that cuff-injury also led to an up-regulated expression of TRPM2. Double immunofluorescent labeling of TRPM2 and α-smooth muscle actin showed a large amount of TRPM2-positive SMCs in the neointimal region. Compared with the normal medial SMCs isolated from non-cuffed arteries, the neointimal SMCs from cuff-injured arteries displayed a greater [Ca²⁺] rise in response to hypoxanthine-xanthine oxidase, which was inhibited by pre-treatment with a TRPM2-specific blocking antibody TM2E3. In mouse carotid arteries, cuff placement for 3 weeks caused clear neointimal formation, accompanied by up-regulated expression of TRPM2. Trpm2 disruption dramatically reduced the neointimal growth. Human saphenous vein samples obtained during CABG surgery were organ-cultured for 2 weeks to allow growth of neointima. Double immunofluorescent labeling of TRPM2 and α-smooth muscle actin showed that the neointima contained numerous TRPM2-positive SMCs. Neointimal hyperplasia in the veins was effectively suppressed by in vitro treatment with TM2E3 or a chemical blocker 2-aminoethoxydiphenyl borate. Furthermore, the effect of TRPM2 blockers and Trpm2 disruption on hydrogen peroxide-induced migration and proliferation of cultured rodent aortic SMCs were evaluated by scratch wound healing assay and MTT assay, respectively. It was found that exposure to hydrogen peroxide for 48 hour substantially enhanced the migration and proliferation of rodent aortic SMCs. In rat aortic SMCs, both TM2E3 and 2-aminoethoxydiphenyl borate significantly inhibited the hydrogen peroxide-induced cell migration and proliferation. The hydrogen peroxide-induced cell migration and proliferation of SMCs was also reduced in Trpm2 knockout mice.<br>Taking together, these results provide strong evidences that in vivo neointimal hyperplasia is accompanied by an up-regulated expression of TRPM2 and that TRPM2 plays a key role in neointimal hyperplasia, VSMCs migration and proliferation. Blocking TRPM2 can be a potential therapeutic approach for protecting blood vessels against intimal thickening.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Ru, Xiaochen.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2013.<br>Includes bibliographical references (leaves 125-151).<br>Abstracts also in Chinese.<br>Declaration of Originality --- p.i<br>Abstract --- p.ii<br>論文摘要 --- p.iv<br>Acknowledgements --- p.vi<br>Abbreviations and Units --- p.vii<br>Table of Content --- p.x<br>List of Figures --- p.xvi<br>List of Tables --- p.xviii<br>Chapter Chapter 1 --- Introduction --- p.1<br>Chapter 1.1 --- Neointimal hyperplasia --- p.1<br>Chapter 1.1.1 --- Definition of neointimal hyperplasia --- p.2<br>Chapter 1.1.2 --- Medical significance of coronary neointimal hyperplasia --- p.3<br>Chapter 1.1.3 --- Pathogenesis of neointimal hyperplasia --- p.5<br>Chapter 1.1.3.1 --- “Response to injury“ hypothesis --- p.6<br>Chapter 1.1.3.2 --- Role of VSMCs --- p.7<br>Chapter 1.1.3.2.1 --- VSMC phenotypic switch --- p.7<br>Chapter 1.1.3.2.2 --- Ca²⁺ channel modulation in VSMCs --- p.8<br>Chapter 1.1.3.2.3 --- VSMC migration --- p.9<br>Chapter 1.1.3.2.4 --- VSMC proliferation --- p.10<br>Chapter 1.1.3.2.5 --- Extracellular matrix production by VSMCs --- p.11<br>Chapter 1.1.3.3 --- Endothelial dysfunction --- p.11<br>Chapter 1.1.3.4 --- Platelet adhesion --- p.12<br>Chapter 1.1.3.5 --- Inflammation --- p.13<br>Chapter 1.1.4 --- Role of ROS in neointimal hyperplasia --- p.14<br>Chapter 1.1.4.1 --- Types of ROS --- p.15<br>Chapter 1.1.4.1.1 --- Superoxide anion --- p.16<br>Chapter 1.1.4.1.2 --- Hydroxyl radical --- p.16<br>Chapter 1.1.4.1.3 --- Hydrogen peroxide --- p.16<br>Chapter 1.1.4.1.4 --- Nitric oxide --- p.17<br>Chapter 1.1.4.2 --- Sources of ROS in vessel wall --- p.17<br>Chapter 1.1.4.3 --- ROS signaling in endothelial cells --- p.19<br>Chapter 1.1.4.4 --- ROS signaling in VSMCs --- p.20<br>Chapter 1.1.4.5 --- ROS and atherosclerosis --- p.21<br>Chapter 1.1.5 --- Current therapeutic approaches to neointimal hyperplasia --- p.23<br>Chapter 1.1.5.1 --- Pharmacological approaches --- p.23<br>Chapter 1.1.5.2 --- Technical Approaches --- p.25<br>Chapter 1.2 --- Transient receptor potential melastatin 2 (TRPM2) channel --- p.27<br>Chapter 1.2.1 --- TRP Channels --- p.27<br>Chapter 1.2.2 --- TRPM2 structure and expression --- p.29<br>Chapter 1.2.2.1 --- Structure --- p.29<br>Chapter 1.2.2.2 --- Alternative splicing isoforms --- p.30<br>Chapter 1.2.2.3 --- Expression pattern --- p.32<br>Chapter 1.2.3 --- TRPM2 channel properties --- p.32<br>Chapter 1.2.4 --- TRPM2 activators and inhibitors --- p.32<br>Chapter 1.2.4.1 --- Activators --- p.33<br>Chapter 1.2.4.1.1 --- ADPR --- p.33<br>Chapter 1.2.4.1.2 --- NAD, cADPR and NAADP --- p.33<br>Chapter 1.2.4.1.3 --- H₂O₂ and oxidative stress --- p.34<br>Chapter 1.2.4.1.4 --- Ca²⁺ --- p.34<br>Chapter 1.2.4.1.5 --- Other regulators --- p.35<br>Chapter 1.2.4.2 --- Inhibitors --- p.35<br>Chapter 1.2.5 --- Biological relevance of TRPM2 --- p.36<br>Chapter 1.2.5.1 --- TRPM2 in insulin release --- p.36<br>Chapter 1.2.5.2 --- TRPM2 in inflammation --- p.36<br>Chapter 1.2.5.3 --- TRPM2 in cell death --- p.37<br>Chapter 1.2.5.4 --- TRPM2-mediated lysosomal Ca²⁺ release --- p.38<br>Chapter 1.2.5.5 --- TRPM2 and cardiovascular diseases --- p.39<br>Chapter Chapter 2 --- Objectives of the Present Study --- p.40<br>Chapter Chapter 3 --- Materials and Methods --- p.42<br>Chapter 3.1 --- Materials --- p.42<br>Chapter 3.1.1 --- Chemicals --- p.42<br>Chapter 3.1.2 --- Media, supplements and other reagents for cell/tissue culture --- p.44<br>Chapter 3.1.3 --- Antibodies --- p.45<br>Chapter 3.1.4 --- Solutions --- p.46<br>Chapter 3.1.4.1 --- Solutions for immunohistochemical and immunocytochemical staining --- p.46<br>Chapter 3.1.4.2 --- solutions for immunoblotting --- p.47<br>Chapter 3.1.4.3 --- Solutions for Genotyping --- p.49<br>Chapter 3.1.4.4 --- Solutions for hematoxylin and eosin (HE) staining --- p.50<br>Chapter 3.1.4.5 --- Solutions for [Ca²⁺]i measurement --- p.51<br>Chapter 3.1.4.6 --- Solutions for IgG purification --- p.51<br>Chapter 3.1.5 --- Animals --- p.51<br>Chapter 3.1.5.1 --- Rat --- p.51<br>Chapter 3.1.5.2 --- Trpm2 knockout mice --- p.52<br>Chapter 3.1.5.3 --- Rabbit --- p.52<br>Chapter 3.1.5.4 --- Ethics --- p.52<br>Chapter 3.1.6 --- Human Tissue --- p.52<br>Chapter 3.2 --- Methods --- p.54<br>Chapter 3.2.1 --- Rodent models of neointimal hyperplasia --- p.54<br>Chapter 3.2.1.1 --- Cuff-induced vascular injury in rat femoral artery --- p.54<br>Chapter 3.2.1.2 --- Cuff-induced vascular injury in mouse carotid artery --- p.54<br>Chapter 3.2.2 --- Genotyping for Trpm2 knockout mice --- p.55<br>Chapter 3.2.2.1 --- Genomic DNA extraction from tail --- p.55<br>Chapter 3.2.2.2 --- Polymerase Chain Reaction (PCR) --- p.55<br>Chapter 3.2.2.3 --- Agarose gel electrophoresis of DNA --- p.56<br>Chapter 3.2.3 --- Human saphenous vein culture and treatment --- p.56<br>Chapter 3.2.4 --- Generation of anti-TRPM2 antibody, TRPM2-specific blocking antibody TM2E3 and preimmune IgG --- p.57<br>Chapter 3.2.5 --- Histological analysis and immunohistochemistry --- p.58<br>Chapter 3.2.6 --- Western blotting --- p.59<br>Chapter 3.2.7 --- Detection of ROS production by dihydroethidium fluorescence --- p.60<br>Chapter 3.2.8 --- Isolation of rodent neointimal and medial smooth muscle cells --- p.60<br>Chapter 3.2.9 --- Culture of rodent aortic smooth muscle cells --- p.61<br>Chapter 3.2.9.1 --- Cell culture --- p.61<br>Chapter 3.2.9.2 --- Cell identification --- p.61<br>Chapter 3.2.10 --- [Ca²⁺]i measurement --- p.62<br>Chapter 3.2.11 --- Cell proliferation assay --- p.63<br>Chapter 3.2.12 --- Cell migration assay --- p.63<br>Chapter 3.2.13 --- Statistical analysis --- p.64<br>Chapter Chapter 4 --- ROS over-production and TRPM2 up-regulation in cuff-induced rodent neointimal hyperplasia --- p.65<br>Chapter 4.1 --- Introduction --- p.65<br>Chapter 4.2 --- Materials and Methods --- p.66<br>Chapter 4.2.1 --- Cuff-induced vascular injury in rat femoral artery --- p.66<br>Chapter 4.2.2 --- Preparation of anti-TRPM2 antibody, TM2E3 and preimmune IgG --- p.66<br>Chapter 4.2.3 --- Histological analysis and immunohistochemistry --- p.66<br>Chapter 4.2.4 --- Western blotting --- p.67<br>Chapter 4.2.5 --- Detection of ROS production --- p.67<br>Chapter 4.2.6 --- Isolation of rat neointimal and medial smooth muscle cells --- p.68<br>Chapter 4.2.7 --- [Ca²⁺]i measurement --- p.68<br>Chapter 4.2.8 --- Statistical analysis --- p.68<br>Chapter 4.3 --- Results --- p.69<br>Chapter 4.3.1 --- Cuff-induced neointimal hyperplasia in rat femoral arteries --- p.69<br>Chapter 4.3.2 --- ROS over-production in neointimal region of cuff-injured rat femoral arteries --- p.69<br>Chapter 4.3.3 --- TRPM2 up-regulation in neointimal region of cuff-injured rat femoral arteries --- p.69<br>Chapter 4.3.4 --- Enhanced [Ca²⁺]i response to HX-XO in rat neointimal smooth muscle cells --- p.70<br>Chapter 4.4 --- Discussion --- p.81<br>Chapter Chapter 5 --- TRPM2 contributes to human and rodent neointimal hyperplasia --- p.86<br>Chapter 5.1 --- Introduction --- p.86<br>Chapter 5.2 --- Materials and Methods --- p.87<br>Chapter 5.2.1 --- Cuff-induced vascular injury in mouse carotid artery --- p.87<br>Chapter 5.2.2 --- Genotyping for Trpm2 knockout mice --- p.87<br>Chapter 5.2.3 --- Organ culture of human saphenous vein --- p.87<br>Chapter 5.2.4 --- Preparation of anti-TRPM2 antibody, TM2E3 and preimmune IgG --- p.88<br>Chapter 5.2.5 --- Histological analysis and immunohistochemistry --- p.88<br>Chapter 5.2.6 --- Western blotting --- p.88<br>Chapter 5.2.7 --- Isolation of mouse neointimal and medial smooth muscle cells --- p.89<br>Chapter 5.2.8 --- [Ca²⁺]i measurement --- p.89<br>Chapter 5.2.9 --- Statistical analysis --- p.90<br>Chapter 5.3 --- Results --- p.90<br>Chapter 5.3.1 --- Cuff-induced neointimal hyperplasia was reduced in Trpm2 knockout mice --- p.90<br>Chapter 5.3.2 --- [Ca²⁺]i response to HX-XO in mouse neointimal smooth muscle cells --- p.90<br>Chapter 5.3.3 --- Inhibiting TRPM2 reduced the neointimal hyperplasia in in vitro cultured human saphenous vein --- p.91<br>Chapter 5.4 --- Discussion --- p.99<br>Chapter Chapter 6 --- Role of TRPM2 in H₂O₂-stimulated migration and proliferation of vascular smooth muscle cells --- p.103<br>Chapter 6.1 --- Introduction --- p.103<br>Chapter 6.2 --- Materials and Methods --- p.104<br>Chapter 6.2.1 --- Culture of rodent aortic smooth muscle cells --- p.104<br>Chapter 6.2.2 --- Immunocytochemistry --- p.104<br>Chapter 6.2.3 --- Genotyping for Trpm2 knockout mice --- p.104<br>Chapter 6.2.4 --- Preparation of anti-TRPM2 antibody, TM2E3 and preimmune IgG --- p.104<br>Chapter 6.2.5 --- [Ca²⁺]i measurement --- p.105<br>Chapter 6.2.6 --- Cell proliferation assay --- p.105<br>Chapter 6.2.7 --- Western blotting --- p.105<br>Chapter 6.2.8 --- Cell migration assay --- p.106<br>Chapter 6.2.9 --- Statistical analysis --- p.106<br>Chapter 6.3 --- Results --- p.106<br>Chapter 6.3.1 --- H₂O₂-induced [Ca²⁺]i rises in rodent aortic smooth muscle cells --- p.106<br>Chapter 6.3.2 --- Role of TRPM2 in H₂O₂-stimulated smooth muscle cell proliferation --- p.107<br>Chapter 6.3.3 --- Role of TRPM2 in H₂O₂-stimulated smooth muscle cell migration --- p.108<br>Chapter 6.4 --- Discussion --- p.118<br>Chapter Chapter 7 --- General Conclusion and Future Work --- p.121<br>Chapter 7.1 --- Concluding remarks --- p.121<br>Chapter 7.2 --- Future work --- p.123<br>Chapter 7.2.1 --- Specific downstream signaling pathway of TRPM2 that mediates ROS-induced VSMC proliferation and migration --- p.123<br>Chapter 7.2.2 --- Involvement of TRPM2 in leukocyte infiltration and inflammation in vascular wall --- p.124<br>References --- p.125<br>List of Publications --- p.152