The effects of oxidatively modified low density lipoprotein (oxLDL) on atherogenesis may be partly mediated by alterations in the production of nitric oxide (NO) by vascular cells. Lipid hydroperoxides (LOOH) and lysophosphatidylcholine (lysoPC) are the major primary products of LDL oxidation. The purpose of this study was to characterize the effects of oxLDL, LOOH and lysoPC on NO production and the expression of inducible nitric oxide synthase (iNOS) gene in lipopolysaccharide (LPS) stimulated macrophages. LDL was oxidized using an azo-initiator 2,2'-azobis (2- amidinopropane) HCl (ABAP) and octadecadienoic acid was oxidized by lipoxygenase to generate 13-hydroperoxyl octadecadienoic acid (13-HPODE). Our study showed that oxLDL markedly decreased the production of NO, the levels of iNOS protein and iNOS mRNA in LPS stimulated macrophages. The inhibition potential of oxLDL on NO production and iNOS gene expression depended on the levels of LOOH formed in oxLDL and was not due to oxLDL cytotoxicity. Furthermore, 13-HPODE markedly reduced NO production and iNOS protein levels, whereas lysoPC showed only slight reduction. The effects of 13-HPODE and lysoPC did not require an acetylated LDL carrier. Our results suggest that 13-HPODE is a much more potent inhibitor of NO production and iNOS gene expression than lysoPC in LPS stimulated RAW264.7 macrophages.

No description available.

No description available.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23802号 / 医博第4848号 / 新制||医||1058(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 永井 洋士, 教授 羽賀 博典, 教授 木村 剛 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The process of TEMPO-mediated oxidation has gained broad usage towards the preparation of highly charged, carboxyl-functionalized polysaccharides. TEMPO-oxidized nanocelluloses (TONc) of high surface charge and measuring 3 to 5 nm in width have been recently prepared from TEMPO-oxidized pulp. This study examines as-produced and surface-hydrophobized TONc as reinforcing additives in cellulosic polymer matrices. In the first part of the work, covalent (amidation) and non-covalent (ionic complexation) coupling were compared as treatment techniques for the hydrophobization of TONc surfaces with octadecylamine (ODA). Subsequently, TONc and its covalently coupled derivative were evaluated as nanofiber reinforcements in a cellulose acetate butyrate (CAB) matrix. The properties of the resulting nanocomposites were compared with those of similarly prepared ones reinforced with conventional microfibrillated cellulose (MFC). It was found that both ionic complexation and amidation resulted in complete conversion of carboxylate groups on TONc surfaces. As a result of surface modification, the net crystallinity of TONc was lowered by 15 to 25% but its thermal decomposition properties were not significantly altered. With respect to nanocomposite performance, the maximum TONc reinforcement of 5 vol % produced negligible changes to the optical transmittance behavior and a 22-fold increase in tensile storage modulus in the glass transition region of CAB. In contrast, hydrophobized TONc and MFC deteriorated the optical transmittance of CAB by ca 20% and increased its tensile storage modulus in the glass transition region by only 3.5 and 7 times respectively. These differences in nanocomposite properties were attributed to homogeneous dispersion of TONc compared to aggregation of both the hydrophobized derivative and the MFC reference in CAB matrix. A related study comparing TONc with MFC and cellulose nanocrystals (CNC) as reinforcements in hydroxypropylcellulose (HPC), showed TONc reinforcements as producing the most significant changes to HPC properties. The results of dynamic mechanical analysis and creep compliance measurements could be interpreted based on similar arguments as those made for the CAB-based nanocomposites. Overall, this work revealed that the use of TONc (without the need for surface hydrophobization) as additives in cellulosic polymer matrices leads to superior reinforcing capacity and preservation of matrix transparency compared to the use of conventional nanocelluloses. / Ph. D.

Während die zellulären Mechanismen der Pathogenese der Arteriosklerose intensiv untersucht worden sind, ist über die Mechanismen, die zu einer bevorzugten Lokalisation arteriosklerotischer Läsionen in bestimmten Gefäßarealen führen, weniger bekannt. Zur Untersuchung dieser Mechanismen wurden Endothelzellen aus menschlichen Koronararterien, einem Gefäßbereich, in dem häufig arteriosklerotische Läsionen beobachtete werden, isoliert und kultiviert. Endothelzellen der Mikrozirkulation menschlicher Herzen wurden unter gleichen Bedingungen kultiviert und die Reaktionen beider Zellarten verglichen. Inkubation der Zellen mit den in Bezug auf die Bildung arteriosklerotischer Plaques besonders pathogenen oxidierten LDL induzierte in makrovaskulären koronaren Endothelzellen eine stärkere Zunahme der PAI-1 Aktivität (182%, p / While the cellular mechanisms of atherosclerosis have been intensively studied, the mechanisms leading to preferential localization of atherosclerotic lesions are less well understood. To further define these mechanisms, endothelial cells from coronary arteries, i.e. vessels with frequent atherosclerotic lesions, were isolated and grown in vitro. In order to compare the reactions of both cell types, endothelial cells derived from microvessels of human hearts were isolated and cultured under identical conditions. Incubation of endothelial cells with oxidized LDL (75 µg/ml protein) induced a significant increase in PAI-1 activity (182 %, p

A aterosclerose é uma doença degenerativa crônica dos vasos, com conseqüências clínicas agudas que incluem o infarto do miocárdio e o acidente vascular cerebral, resultantes geralmente da ruptura da placa e trombose. É atualmente reconhecida como de característica inflamatória, iniciada e propagada no contexto da hipercolesterolemia. Um trabalho de nosso grupo utilizou técnicas de phage display para comparar placas ateroscleróticas e carótidas normais objetivando a busca de proteínas alteradas potencialmente envolvidas na patogênese da doença. Diversas semaforinas e plexinas (receptores de semaforinas) foram identificadas dentre elas a plexina B1, que possui alta afinidade por CD100, sugerindo assim uma concentração aumentada de CD100 na placa aterosclerótica. CD100 foi a primeira semaforina descrita no sistema imune e a única até hoje descrita como possuidora de duas formas de funcionalidades distintas, sendo uma de membrana (mCD100) e outra solúvel (sCD100). Neste trabalho demonstramos a expressão da semaforina CD100 em macrófagos e células espumosas em placas ateroscleróticas humanas, assim como seu padrão de expressão ao longo da diferenciação monócito-macrófago-célula espumosa, e sob estímulos distintos. Além disso, identificamos pela primeira vez o receptor que medeia suas atividades nessas células, a plexina B2. Adicionalmente, detectamos também pela primeira vez detectamos a expressão de CD100 em células endoteliais teciduais e cultivadas in vitro, o que sugere um papel significativo da semaforina em fenômenos vasculares. Com base nessas observações e nos resultados de experimentos de bloqueio de adesão constatamos que CD100 pode atuar na fase mais precoce da aterosclerose, como uma molécula de adesão envolvida na ligação entre monócitos e células endoteliais. Verificamos ainda que CD100 diminui a captação de LDLox em macrófagos e células espumosas. Poucos estudos relatam a presença ou possível atividade biológica de CD100 tanto na aterosclerose quanto em macrófagos. Devido às já estabelecidas ações no sistema imune, acreditamos que a expressão diferencial dessa semaforina desempenha um papel amplificador na patogênese da aterosclerose. Posteriormente, essa proteína poderá servir como alvo de inibição da progressão da doença e de suas complicações / Atherosclerosis is a chronic degenerative disease affecting vessels, with acute clinical consequences that include myocardium infarction or stroke, generally resulting from plaque rupture and thrombosis. It is now recognized as an inflammatory disease, initiated and developed in a hipercholesterolemic context. A work in our lab has used phage display techniques to compare atherosclerotic plaques and normal carotids, searching for altered proteins potentially involved in the pathogenesis of the disease. Many semaphorins and plexins (semaphorin receptors) have been identified, among which plexin B1, a high affinity receptor for CD100, suggesting an augmented level of CD100 in the atherosclerotic plaques. CD100 is the first semaphorin described in the immune system, and the only to possess two forms with distinct functionalities, being one associated to the membrane, mCD100, and another soluble form, sCD100. In the present work we have demonstrated CD100 expression in macrophages and foam cells of human atherosclerotic plaques, as well as its pattern of expression along monocyte-macrophage-foam cell differentiation and under distinct stimuli. Furthermore, we have identified for the first time the receptor involved in CD100 activities in these cells, namely plexin B2. Aditionally, we have detected CD100 expression in tissue as well as in in vitro cultured endothelial cells, also for the first time. According to these informations and adhesion blockage experiments we have shown that CD100 may act in the earliest phase of the establishment of atherosclerosis, as an adhesion molecule involved in monocyte-endothelial cell association. We have also verified that CD100 diminishes the intake of oxLDL in macrophages and foam cells. Only a few studies describe the presence or possible biological activity of CD100 in atherosclerosis or macrophages. Since the molecule has been shown to participate in the immune system, we believe that the differential expression of this semaphorin plays an amplifying role in the pathogenesis of atherosclerosis. In the future, this protein could act as an inhibition target of the disease progression as well as its complications

A aterosclerose é uma doença degenerativa crônica dos vasos, com conseqüências clínicas agudas que incluem o infarto do miocárdio e o acidente vascular cerebral, resultantes geralmente da ruptura da placa e trombose. É atualmente reconhecida como de característica inflamatória, iniciada e propagada no contexto da hipercolesterolemia. Um trabalho de nosso grupo utilizou técnicas de phage display para comparar placas ateroscleróticas e carótidas normais objetivando a busca de proteínas alteradas potencialmente envolvidas na patogênese da doença. Diversas semaforinas e plexinas (receptores de semaforinas) foram identificadas dentre elas a plexina B1, que possui alta afinidade por CD100, sugerindo assim uma concentração aumentada de CD100 na placa aterosclerótica. CD100 foi a primeira semaforina descrita no sistema imune e a única até hoje descrita como possuidora de duas formas de funcionalidades distintas, sendo uma de membrana (mCD100) e outra solúvel (sCD100). Neste trabalho demonstramos a expressão da semaforina CD100 em macrófagos e células espumosas em placas ateroscleróticas humanas, assim como seu padrão de expressão ao longo da diferenciação monócito-macrófago-célula espumosa, e sob estímulos distintos. Além disso, identificamos pela primeira vez o receptor que medeia suas atividades nessas células, a plexina B2. Adicionalmente, detectamos também pela primeira vez detectamos a expressão de CD100 em células endoteliais teciduais e cultivadas in vitro, o que sugere um papel significativo da semaforina em fenômenos vasculares. Com base nessas observações e nos resultados de experimentos de bloqueio de adesão constatamos que CD100 pode atuar na fase mais precoce da aterosclerose, como uma molécula de adesão envolvida na ligação entre monócitos e células endoteliais. Verificamos ainda que CD100 diminui a captação de LDLox em macrófagos e células espumosas. Poucos estudos relatam a presença ou possível atividade biológica de CD100 tanto na aterosclerose quanto em macrófagos. Devido às já estabelecidas ações no sistema imune, acreditamos que a expressão diferencial dessa semaforina desempenha um papel amplificador na patogênese da aterosclerose. Posteriormente, essa proteína poderá servir como alvo de inibição da progressão da doença e de suas complicações / Atherosclerosis is a chronic degenerative disease affecting vessels, with acute clinical consequences that include myocardium infarction or stroke, generally resulting from plaque rupture and thrombosis. It is now recognized as an inflammatory disease, initiated and developed in a hipercholesterolemic context. A work in our lab has used phage display techniques to compare atherosclerotic plaques and normal carotids, searching for altered proteins potentially involved in the pathogenesis of the disease. Many semaphorins and plexins (semaphorin receptors) have been identified, among which plexin B1, a high affinity receptor for CD100, suggesting an augmented level of CD100 in the atherosclerotic plaques. CD100 is the first semaphorin described in the immune system, and the only to possess two forms with distinct functionalities, being one associated to the membrane, mCD100, and another soluble form, sCD100. In the present work we have demonstrated CD100 expression in macrophages and foam cells of human atherosclerotic plaques, as well as its pattern of expression along monocyte-macrophage-foam cell differentiation and under distinct stimuli. Furthermore, we have identified for the first time the receptor involved in CD100 activities in these cells, namely plexin B2. Aditionally, we have detected CD100 expression in tissue as well as in in vitro cultured endothelial cells, also for the first time. According to these informations and adhesion blockage experiments we have shown that CD100 may act in the earliest phase of the establishment of atherosclerosis, as an adhesion molecule involved in monocyte-endothelial cell association. We have also verified that CD100 diminishes the intake of oxLDL in macrophages and foam cells. Only a few studies describe the presence or possible biological activity of CD100 in atherosclerosis or macrophages. Since the molecule has been shown to participate in the immune system, we believe that the differential expression of this semaphorin plays an amplifying role in the pathogenesis of atherosclerosis. In the future, this protein could act as an inhibition target of the disease progression as well as its complications

Apoptosis plays a crucial role in multicellular organisms by preserving tissue homeostasis and removing harmful cells. The anti-apoptotic B-cell CLL/lymphoma 2 (Bcl-2) and the pro-apoptotic Bcl-2-associated X protein (Bax) act as major regulators of the mitochondrial apoptotic pathway. Activation of Bax via stress signals causes its translocation to the mitochondrial outer membrane (MOM). There, Bax forms homo-oligomeric pores, leading to the release of apoptogenic factors, caspase activation and ultimately cell death. However, the underlying mechanism for the recruitment and pore forming activity of Bax is still not elucidated. Nevertheless, the mitochondrial membrane system seems to play an active and crucial role, presumably being directly involved in the onset of the mitochondrial apoptosis. Since the formation of reactive oxygen species (ROS) is a common stress signal and one of the hallmarks of the mitochondrial apoptosis, direct damage can occur to these membranes by the generation of oxidized phospholipids (OxPls), whose presence can crucially influence the pro-apoptotic action of Bax there. To better understand the impact of OxPls on membranes as well as their potential role in the mitochondrial apoptotic process, defined OxPl species were incorporated into phospholipid vesicles and studied with various biophysical techniques. Differential scanning calorimetry (DSC) and solid state nuclear magnetic resonance (NMR) spectroscopy were used to gain insight into changes in membrane properties in the presence of OxPls. In addition to circular dichroism (CD) spectroscopy, DSC and solid state NMR were furthermore performed to elucidate the impact of OxPls on Bax-membrane interactions. The occurrence of OxPls gave rise to dramatic changes in membrane organization and dynamics, manifested as lateral phase separation into OxPl-rich and -poor domains and modified hydration at the membrane interface. The presence of OxPls also had a great impact on the interaction between Bax and mitochondria-mimicking vesicles, strongly promoting the association of the protein with the membrane. At the MOM, Bax is believed to be inhibited by Bcl-2. How this inhibition occurs is still a mystery due to the lack of biophysical information on Bcl-2, in particular on the full-length protein variant. Since Bcl-2 is also one of the main culprits in the progression of various forms of cancer, knowledge of the structural and mechanistic properties of the full-length protein is essential for a fundamental understanding of its function at a molecular level. To this end, a method for the production of full-length Bcl-2 was developed. By performing cell-free protein synthesis, preparative amounts of the protein were obtained, which enabled a biophysical characterization of the putative interaction between Bax and Bcl-2 using CD and fluorescence spectroscopy. A protocol for the reconstitution of Bcl-2 into proteoliposomes was also developed, promising for future studies of the full-length protein in its native membrane environment; a prerequisite to fully understand its pro-survival functions as well as providing crucial information for the design of novel anti-cancer drugs.

Introduction: Fast and convenience foods are abundant, relatively inexpensive, and accommodating to the fast-paced lifestyle of many Americans. One popular method of cooking used by many fast food establishments is deep-fat frying. Soybean oil is commonly used for frying and is rich in polyunsaturated fatty acids (PUFA) such as linoleic acid (LA). When soybean oil is used for deep-fat frying, LA becomes oxidized (Ox-LA). Endogenous Ox-LA has the capacity to be a ligand to peroxisome proliferator-activated receptor gamma (PPAR¥ã), a nuclear transcription factor that regulates adipocyte maturation. It is not yet known whether or not dietary Ox-LA has the same capacity with respect to PPAR¥ã. Considering the fact that dietary oxidized lipids are abundant in the typical American diet, it is important to know if they regulate weight gain and especially adipose tissue mass. In this study, we investigate the effects of fresh and heated soybean oil on weight gain and adiposity in mice fed isocaloric low fat diets. Methods: Soybean oil was heated on a hot plate, under a hood, at 190¨¬C for three hours. Fresh soybean oil served as the source of unoxidized oil (Unox-oil) and the heated oil served as the source of oxidized oil (Ox-oil). Both the Ox-oil and Unox-oil were incorporated into a low-fat (10% of calories) mouse chow by Research Diets, Inc. (New Brunswick, NJ). Sixteen C57BL/6J mice were divided into two groups and fed low fat diets with Ox-oil (low fat oxidized, LFO) or with Unox-oil (low fat unoxidized, LFU). Another group of 8 mice were pair fed to the LFO group with the Unox-oil containing chow (PLU). Mice in the LFO and LFU groups were fed ad libitum and known amounts of fresh food was added to the cages every three days. Leftover food was weighed. Body weights were measured once a week. After 16 weeks mice were euthanized and epididymal white adipose tissue (EWAT), retroperitoneal white adipose tissue (RWAT), inguinal white adipose tissue (IWAT), and intrascapular brown adipose tissue (IBAT) samples were collected, weighed and stored at -80 degrees Celsius until further analysis. Fat pads were homogenized and cytosolic and nuclear proteins were extracted by standard methods. These extracts were subjected to Western blotting to determine the amount of PPAR¥ã in the cytosol and nuclear compartments of the fat pads. Differences in group means were analyzed by Mann Whitney U test. Comparisons were considered statistically significant at a p-value of < 0.05. Results: Final mean body weights were significantly different when comparing the mice in the LFU group to the pair fed mice (PLU) (mean ¡¾ SD; 29.52 ¡¾ 1.09 grams (g) and 26.85 ¡¾ 1.44 g, respectively; p < 0.05). Mice fed a low fat diet consisting of Ox-oil (LFO) had a final mean body weight of 27.88 ¡¾ 2.03 g. Mice in the LFU group gained significantly more weight on average than did mice in the LFO or PLU groups (mean ¡¾ SD; 8.86 ¡¾ 1.37g, 7.10 ¡¾ 1.47 g, and 5.71 ¡¾ 1.13 g, respectively). Although mean food intakes were not significantly different between any of the three groups, the average food intake was greatest for the LFU mice in comparison to the LFO and the PLU mice (mean ¡¾ SD; 20.65 ¡¾ 0.09 g/week, 18.40 ¡¾ 0.05 g/week, and 18.38 ¡¾ 0.19 g/week, respectively). Feeding efficiency (g of weight gain/g of food consumed) was the highest in the LFU mice compared to the PLU mice (mean ¡¾ SD; 0.031 ¡¾ 0.005 g/g and0.022 ¡¾ 0.004 g/g) and this difference was statistically significant. The LFO mice gained less weight per gram of food consumed than did the LFU mice (mean ¡¾ SD; 0.028 ¡¾ 0.006 g/g). Mean weights of all fat pads in the LFO group were significantly greater than those of the LFU and PLU mice (mean ¡¾ SD; 0.329 ¡¾ 0.109g, 0.199 ¡¾ 0.055g, and 0.219 ¡¾ 0.041 for EWAT, 0.091 ¡¾ 0.039g, 0.050 ¡¾ 0.026g, and 0.051 ¡¾ 0.017 for RWAT, 0.221 ¡¾ 0.065g, 0.135 ¡¾ 0.053g, and 0.144 ¡¾ 0.038 for IWAT, and 0.079 ¡¾ 0.012g, 0.055 ¡¾ 0.013g, and 0.062 ¡¾ 0.011 for IBAT, respectively). PPAR¥ã protein in the cytosol of EWAT fat pads was analyzed and quantified in comparison to the amount of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; loading control) present. Mean PPAR¥ã /GAPDH ratios for LFU mice was 0.226 ¡¾ 0.082, for LFO mice was 0.264 ¡¾ 0.122, and for PLU mice was 0.234 ¡¾ 0.108. Mean PPAR¥ã:GAPDH ratios were not significantly different between any of the groups. Conclusion: It appears that the consumption of oxidized oil caused a significant decrease in weight gain and food intake (although not significant) and a significant increase in fat pad mass in mice compared to those consuming a diet with unoxidized oil. The lack of difference in the amount of PPAR¥ã among the three groups of mice suggests that the changes in weight gain and fat pad mass among the oxidized oil consuming animals is not mediated through regulation of PPAR¥ã protein. To our knowledge, ours is the first study to report that mice consuming a low fat diet inclusive of dietary oxidized lipids exhibit greater adiposity than do mice consuming a low fat diet consisting of unoxidized lipids.