Thesis (Ph. D.)--University of Sydney, 2002. / Title from title screen (viewed Apr. 28, 2008). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Molecular and Microbial Biosciences, Faculty of Science. Includes bibliographical references. Also available in print form.

O objetivo deste estudo foi investigar os efeitos do hipotiroidismo experimental sobre o leito vascular da aorta torácica. Para a análise histológica foram realizadas colorações de hematoxilina-eosina, picrosirius e Weigert. Na análise de expressão proteica, foram realizadas as quantificações para colágeno I e III, elastina, MMP-9 e MMP-2, TIMP-1 e TIMP-2. As análises histológicas demonstraram uma diminuição da AST das aortas dos animais hipo e juntamente a esta alteração, foi constatada a diminuição da expressão proteica de colágeno do tipo I. Em relação à elastina, foi possível observar aumento da expressão deste elemento nas aortas dos animais hipotiroideos. Na avaliação da expressão proteica para MMP-9, foi possível verificar uma redução desta proteína no grupo hipotiroideo, assim como um aumento da expressão de TIMP-2. Frente aos presentes resultados, é possível sugerir que o estado hipometabólico desencadeado pelo hipotiroidismo afeta as CMLVs comprometendo mecanismos de síntese/degradação, alterando o importante arranjo da MEC presente na aorta torácica. / The aim of this study was to investigate hypothyroidism effects on thoracic aorta wall. For histological analyses were performed stains like hematoxilin-eosin, picrosirius and Weigert. In the protein expression assays were performed quantification for collagen I and III, elastin, MMP-9, MMP-2, TIMP-1 and TIMP-2. The histological analyses showed a decrease in aortas CSA and also a decrease in protein expression of collagen I in the hypo group. As regards to elastin, was possible to see an increase of this protein expression in hypo animals. In the evaluation for MMP-9 expression, was found a decrease in this protein and for TIMP-2 an increase in hypothyroidism group. Facing to these results, is possible to suggest that the hypometabolic state triggered by hypothyroidism, affects the VSMCs compromising mechanisms of synthesis/degradation and changing the important constitution of thoracic aorta ECM.

Abstract Numerous adverse effects and health problems are associated with smoking, but the mechanisms of the adverse effects of smoking on skin are not well documented. The aim of the present study was to elucidate the effects of smoking on the structure, metabolism and appearance of skin. The study population consisted of 98 Finnish males, of whom 47 were current smokers and 51 non-smokers. The main parameters under evaluation were the appearance and physical qualities of skin, including skin wrinkling, thickness and elasticity. Biochemical analyses were performed to assess the rate of type I and III collagen biosynthesis as well as the degradation of the extracellular matrix (ECM) of skin in terms of matrix metalloproteinase levels (MMPs). To compare the morphology of skin between the groups, histological and immunohistological studies were performed, including assessments of the proportional area and width of dermal elastic fibres. The results revealed decreased synthesis of type I and III collagens in smokers as well as changes in the regulatory mechanisms which control the turnover of these and other extracellular matrix proteins. The level of matrix metalloproteinase -8 (collagenase-2), a protease degrading both type I and type III collagen, in suction blister fluid was significantly higher in smokers, indicating enhanced degradation of these collagens. In skin tissue samples, the levels of the active forms of MMP-8 and MMP-9 were significantly lower in smokers compared to non-smokers. Serum levels of MMP-8 were slightly but not significantly higher in smokers, whereas the levels of the matrix metalloproteinases MMP-2 and MMP-9 (72-kDa and 92-kDa gelatinase, respectively) were significantly higher in smokers compared to non-smokers. Salivary MMP-8 and MMP-9 were lower in smokers compared to non-smokers, but only the latter showed a statistically significant difference. The levels of the tissue inhibitor of matrix metalloproteinases (TIMP-1) were significantly lower in the suction blister fluid of smokers compared to non-smokers. In general, there were no significant differences in skin thickness and elasticity or regeneration of barrier function, nor in the amount or width of elastic fibres between the groups. We did not observe significant differences in skin wrinkling between smokers and non-smokers, but smokers looked older than their age compared to non-smokers. It can be concluded that the rate of type I and III collagen synthesis in skin is decreased and the regulation of ECM turnover is altered in smokers, which may lead to deterioration of the tensile strength and resiliency of skin in the long term, even though no morphological changes were detected in the present study.

Chronic changes in cardiac load can cause the vertebrate heart to remodel. For ectotherms, ambient temperature can directly alter cardiac load. Therefore, long-term ambient temperature change can initiate a dynamic cardiac remodelling response to preserve cardiac function. The aims of my PhD thesis were to study the effects of chronic temperature change on the ectothermic heart and cardiovascular system, using the cold-active rainbow trout and the cold-dormant freshwater turtle. In contrast to the majority of previous studies, my experiments focused on the passive, rather than active, properties of the heart. In results chapters 3, 4, 5 and 6, I studied the effects of thermal remodelling on the rainbow trout heart. Chronic cold caused a global increase in chamber stiffness, both at the whole chamber and micromechanical level, with an associated myocardial fibrosis. In the ventricle and atrium there was an up-regulation of collagen promoting genes. In the ventricle, I found cold-induced hypertrophy of the spongy myocardium with an up-regulation of hypertrophic growth factors, which was associated with an increase in tissue lipid suggesting an increase in fatty acid oxidation (FAO). In the atrium, there was no hypertrophy, but there was an increase in extra-bundular sinus, suggesting chronic dilation. Chronic warming initiated an opposite response, with increased cardiac compliance associated with an up-regulation of collagen degrading genes in the ventricle and atrium. In the outflow tract (OFT) and atrium, this increased activity of matrix metalloproteinase (MMPs) and in the OFT abundance of MMPs was increased. The warmed ventricle showed atrophy of the spongy myocardium with a decrease in lipid and an increase in glycogen suggesting a switch in cellular energetics from FAO to glycolytic pathways. In chapters 7, 8 and 9, I studied the effects of thermal remodelling on the freshwater turtle heart. I found an in vivo decrease in systemic resistance causing an increased right to left cardiac shunt flow, associated with an increased elastin content of the major outflow vessels. Cold acclimation increased cardiac sensitivity to preload as well as whole chamber passive stiffness and micromechanical stiffness of tissue sections, associated ventricular fibrosis and increased collagen coherency. In addition, chronic cold decreased the gelatinase activity of MMPs and increased mRNA expression of a tissue inhibitor of MMPs. Furthermore, chronic cold was associated with a decrease in tissue lipid and phosphates, but an increase in tissue protein, glycogen and lactate. These changes in tissue biochemistry suggest a switch in cellular energetics from FAO to glycolytic pathways, likely due to the decreased oxygen availability associated with winter inactivity. Overall, the chambers of the ectothermic heart show distinct remodelling phenotypes, which likely reflect their in cardiac function. Thermal remodelling of the fish ventricle serves both cardio-protection, from the haemodynamic strain of changes in cardiac preload and afterload, as well as compensation for the direct effects of temperature. In the turtle, changes in compliance and cellular energetics of the ventricle suggest a cardio-protective mechanism preparing the heart for increased haemodynamic stress and hypoxic or anoxic conditions during inactive winter hibernation.

Abstract Lysyl oxidases (EC 1.4.3.13, protein-lysine 6-oxidases) are extracellular copper enzymes that initiate the cross-linking of collagens and elastin by catalyzing oxidative deamination of the ε-amino group in certain lysine and hydroxylysine residues. The cross-links formed are responsible for the tensile strength of collagen fibers and the unique elastic properties of elastin. Three human lysyl oxidase isoenzymes, lysyl oxidase (LOX), lysyl oxidase-like protein (LOXL), and lysyl oxidase-like 2 protein (LOXL2), have been identified and characterized so far. Two additional human lysyl oxidase isoenzymes, lysyl oxidase-like 3 (LOXL3) and lysyl oxidase-like 4 (LOXL4), proteins were identified, cloned, and partially characterized in this study. Both polypeptides showed a high degree of overall similarity to each other and to the LOXL2 polypeptide, whereas the two polypeptides showed a significant similarity to LOX and LOXL only in the C-terminal region, which contains all amino acid residues thought to be needed for the catalytic activity of the LOX enzyme. The LOXL3 gene is expressed in several tissues, the highest expression levels being in the placenta, heart, ovary, testis, small intestine, and spleen. The LOXL4 gene is likewise expressed in most human tissues studied, the highest levels being seen in the skeletal muscle, testis, and pancreas. Both polypeptides were shown to be secreted extracellular proteins. The role of the first described LOX isoenzyme was studied by inactivating its gene in mice. Most Lox-/- embryos died at the end of gestation, and the few live-born pups were cyanotic and died within a few hours, autopsy revealing large aortic aneurysms. Light microscopy demonstrated structural abnormalities in the aortic walls of Lox-/- embryos, and further analysis by electron microscopy showed highly fragmented elastic fibers, discontinuity in the smooth muscle cell layers, and endothelial cell damage. Doppler ultrasonography of Lox-/- embryos in utero revealed multiple signs of cardiovascular dysfunction, which contributed to the early death of the Lox-/- mice. The results indicate that Lox has an essential role in the development and function of the cardiovascular system and that this role cannot be replaced to any significant extent by other lysyl oxidase isoenzymes.

La maladie cardiovasculaire la plus courante dans les pays industrialisés est la maladie coronarienne responsable d’une ischémie du tissu cardiaque pouvant conduire à l’infarctus du myocarde. Bien que les améliorations de la prise en charge aient considérablement réduit les délais de la reperfusion (seul remède à l’ischémie), l’ischémie/reperfusion (I/R) entraîne des dommages cellulaires et tissulaires ainsi qu’une diminution des capacités fonctionnelles du coeur. Il existe néanmoins des systèmes de cardioprotection endogènes (comme le préconditionnement (préC) ou le postconditionnement (postC)) que l’on peut activer via l’administration de substances pharmacologiques. L’élastine, une protéine fibreuse de la matrice extracellulaire, est responsable de l’élasticité de certains de nos tissus (poumons, peau, <). Des peptides issus de la dégradation de l’élastine (PE) exercent un effet cardioprotecteur envers l’I/R en activant la voie de survie cellulaire RISK. Dans des fibroblastes dermiques, la transduction du signal induit par les peptides d’élastine est médiée de façon précoce par un second messager : le lactosylcéramide (LacCer). Cette étude constitue une première approche des effets du LacCer dans la cardioprotection envers l’I/R. Nous avons montré que le LacCer est un médiateur précoce du signal cardioprotecteur induit par les PE et qu’il protège le coeur des dommages liés à l’I/R dans un modèle de coeur isolé et perfusé en postC. Lors du vieillissement, de nombreuses modifications physiologiques sont à l’origine d’une perte d’efficacité des mécanismes de cardioprotection. Dans ce travail, nous avons également étudié les effets cardioprotecteurs des PE, du LacCer et de l’inhibition de p38MAPK (une protéine probablement impliquée dans la perte des mécanismes cardioprotecteurs liée au vieillissement) envers les dommages liés à l’I/R chez les rats âgés. Nous avons montré que les PE exercent un effet cardioprotecteur en pré+postC contre l’I/R au niveau de la survie cellulaire et au niveau de la récupération des capacités contractiles cardiaques. Le LacCer n’exerce un effet protecteur qu’au niveau de la survie cellulaire et l’inhibition de p38MAPK entraine une meilleure récupération des capacités contractiles sans améliorer la survie cellulaire. / In developed countries the most common cardiovascular disease is coronary heart disease that is responsible for myocardial ischemia and can lead to myocardial infarction. Reperfusion is the only cure for ischemia. Care improvement has dramatically reduced reperfusion delay but ischemia/reperfusion (I/R) causes a lot of cellular and tissular damages and a reduction of cardiac contractile abilities. Nevertheless cardioprotective pathways (like preconditionning (preC) and postconditionning (postC) can be pharmacologically triggered to reduce I/R injury. Elastin is a fibrous protein from extracellular matrix and is responsible for tissues elasticity in lungs, skin, < Peptides derived from elastin fragmentation (EP) exhibit cardioprotective function against I/R by triggering the RISK pathway, a cell survival pathway. In dermic fibroblasts, elastin peptides pathway is mediated by an early messenger : lactosylcéramide (LacCer). In this work we have studied cardioprotective function of LacCer against I/R injury. We show that LacCer is an early messenger of EP cardioprotective pathway and that it also exhibits a cardioprotective function against I/R in an postconditionning isolated rat heart model. During ageing dramatic physiological modifications are responsible for a loss of efficiency of cardioprotection pathways. In this work we have also studied the potential cardioprotective function of EP, LacCer and p38MAPK inhibition (a protein presumably involved in loss of efficiency of cardioprotection pathways during ageing) against I/R injury in aged rats. We have shown that EP exhibit a cardioprotective function against I/R injury in aged rats as well as young rats in a pre+postconditionning protocol. EP enhance contractile abilities recovering and cell survival. LacCer improves only cell survival and p38MAPK inhibition improves only contractile abilities recovering

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20236号 / 医博第4195号 / 新制||医||1019(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 木村 剛, 教授 松原 和夫, 教授 川村 孝 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

In cardiac biomechanics, there is an apparent knowledge gap in 3D cardiac elastin structure and its biomechanical roles. In this study, we fill this knowledge gap via novel biomedical imaging and bioengineering means. In Aim 1, we created an overall mapping of 3D microstructures of the epicardial elastin fibers on porcine left ventricles (LV) using a laser scanning confocal microscope. We demonstrated the location- and depth-dependencies of the epicardial elastin network. Histological staining was also applied to reveal the patterns of endocardial and interstitial elastin fibers, as well as elastin fibers associated with the Purkinje fibers. In Aim 2, a novel algorithm was developed to better reconstruct the elastin fiber network and extract topological fiber metrics. We created a “fiberness” mask via fiber segmentation and fiber skeletonization to obtain the one-voxel-thick centerline skeleton and remove spurious fiber branches, thus generating topological and geometrical descriptors and bringing the study of cardiac elastin to a new level. In Aim 3, we successfully developed a semi-quantitative approach to characterize the residual stress in the epicardial layer by calculating the total angular change due to curling. Our novel curling angle characterization clearly reveals the existence of residual stress as well as the direction (circumferential vs. longitudinal) and location-dependency of the residual stress. In Aim 4, for the first time we estimated the regional residual stress of the epicardial layer on the intact LV via a four-step methodology: (i) quantify regional residual strains by comparing in situ and stressree marker dimensions; (ii) obtain regional tension-stretch/stress-stretch curves along the circumferential and longitudinal directions; (iii) adjust the biaxial curves to the 0g load reference; (iv) estimate the circumferential and longitudinal residual stresses via residual strains. This method accurately estimates the residual stress in the epicardial layer in various LV anatomical locations. We found that the location-dependency of circumferential and longitudinal residual stresses correlates with the curvature of heart surfaces. Our studies show that the epicardial layer, with its rich elastin content, might function as a balloon that wraps around the heart, and the residual stress sets up a boundary condition that assists with LV contraction.

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yes / Polypropylene mesh for pelvic floor surgery is associated with serious complications ( e.g. erosion). A biodegradable tissue engineered mesh composed of a polylactic acid (PLA) scaffold seeded with autologous cells is a promising alternative. However, thus far elastin content (important for elastic recoil) in this tissue has been low. We aimed to increase elastin expression and test the resultant tensile properties.