Chronic intake of alcohol can result in a range of pathology in the liver. Whilst the earliest changes observed with chronic ethanol, including the accumulation of lipid, or steatosis, are readily reversible upon cessation of alcohol consumption, longer exposure to ethanol may achieve more complex disease states including steatohepatitis, fibrosis, and cirrhosis that can cause irreversible damage and progress to fulminant hepatic failure. A key concept in the pathogenesis of alcoholic liver disease is that chronic ethanol primes the liver to increased injury through an interplay between hepatocytes and non-parenchymal cells, chiefly immune cells, of the liver. These relationships between hepatocytes and non-parenchymal cell types in alcoholic liver disease are reviewed in Chapter 1A. The Hypoxia Inducible Factors are a set of transcription factors that classically have been described as affecting a homeostatic response to conditions of low oxygen tension. Alcoholic liver disease is marked by increased hepatic metabolic demands, and some evidence exists for increased hepatic tissue hypoxia and upregulation of hypoxia-inducible factor mRNA with chronic alcohol. However, the biological significance of these findings is unknown. In Chapter 1B, we review the literature on recent investigations on the role of hypoxia inducible factors in a broad array of liver diseases, seeking to find common themes of biological function. In subsequent chapters, we investigate the hypothesis that a member of the hypoxia inducible- factor family, HIF1α, has a role in the pathogenesis of alcoholic liver disease. In Chapter 2, we establish a mouse model of alcoholic liver disease and report data confirming HIF1α activation with chronic ethanol. We demonstrate that HIF1α protein, mRNA, and DNA binding activity is upregulated in ethanol-fed mice versus pair-fed mice, and that some upregulation of HIF2α protein is observable as well. In Chapter 3, we utilize a mouse model of hepatocyte-specific HIF1α activation and demonstrate that such mice have exacerbated liver injury, including greater triglyceride accumulation than control mice. Using cre-lox technology, we introduce a degradation resistant mutant of HIF1α in hepatocytes, and after four weeks of ethanol feeding, we demonstrate that mice with the HIF1α transgene have increased liver-weight to body weight ratio and higher hepatic triglyceride levels. Additionally, several HIF1α target genes are upregulated. In Chapter 4, we examine the relationship between HIF1α activation and hepatic lipid accumulation using a recently published in vitro system, in which lipid accumulation was observed after treating Huh7 cells with the chemokine Monocyte Chemoattractant Protein-1 (MCP-1). We report that MCP-1 treatment induces HIF1α nuclear protein accumulation, that HIF1α overexpression in Huh7 cells induces lipid accumulation, and finally, that HIF1α siRNA prevents MCP-1 induced lipid accumulation. In Chapter 5, we use mouse models to investigate the hypothesis that suppression of HIF1α in hepatocytes or cells of the myeloid lineage may have differing effects on the pathogenesis of alcoholic liver disease. We find that ethanol-fed mice expressing a hepatocyte-specific HIF1α deletion mutant exhibit less elevation in liver-weight body ratio and diminished hepatic triglycerides versus wild-type mice; furthermore, we find that challenging these mice with lipopolysaccharide (LPS) results in less liver enzyme elevation and inflammatory cytokine secretion than in wild-type mice. In Chapter 6, we offer a final summary of our findings and some directions for future work.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第18927号 / 薬科博第41号 / 新制||薬||5(附属図書館) / 31878 / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 掛谷 秀昭, 教授 大野 浩章, 教授 高須 清誠 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21257号 / 医博第4375号 / 新制||医||1029(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 増永 慎一郎, 教授 妹尾 浩, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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By undertaking a number of different experimental approaches at the genetic, cellular/ molecular and integrative physiology levels, I investigated functional variation in the Hypoxia-Inducible Factor (HIF) transcription pathway in humans. My studies focused on Tibetan natives. Tibetan highlanders are adapted to life in a hypoxic environment and exhibit distinct physiological traits at high altitude. Recent studies identified positive selection at two genetic loci, EPAS1 (HIF2α) and EGLN1 (PHD2), in Tibetan highlanders and demonstrated an association of EGLN1/EPAS1 genotype with haemoglobin concentration. Both are genes of the HIF pathway, which coordinates an organism’s response to hypoxia. Patients living at sea level with genetic diseases of the HIF pathway have characteristic phenotypes at both the integrative physiology and cellular levels. I investigated whether Tibetans living at sea level also possess distinct phenotypic characteristics, and whether these may be related to underlying variation within the HIF pathway. I compared Tibetans living at sea level with Han Chinese, their most closely-related major ethnic group, and found that Tibetans possess a significantly different integrative physiology phenotype. Tibetans had a lower haemoglobin concentration and haematocrit, a higher pulmonary ventilation relative to metabolism, and blunted pulmonary vascular responses to both acute (minutes) and sustained (8 hours) hypoxia. Regarding genotype- phenotype relationships within the Tibetans, I found a significant correlation between both EPAS1 and EGLN1 genotype and the induction of erythropoietin by systemic hypoxia. At an intermediate cellular level, the relative expression and the hypoxic induction of HIF- regulated genes were significantly lower in peripheral blood lymphocytes from Tibetans compared with Han Chinese. I also investigated whether the genetic variation in EPAS1 selected for in Tibetans may be functional at the molecular level by affecting transcription of EPAS1 in cells and whether certain coding variants in <e,>EGLN1 found in Tibetans affect protein (PHD2) activity in cells and in vitro. A small supplementary study was undertaken in patients with idiopathic erythrocytosis, who have elevated or inappropriately normal erythropoietin levels, to investigate if they have genetic alterations in the HIF system.

Adaptation to hypoxia is a well-known phenomenon increasing myocardial resistance to ischemia-reperfusion (I/R) injury as an appropriate physical exercise which improves the contractile function of the heart. Lactate is a major energy substrate for the heart muscle during physical activity and hypoxia. The metabolism of lactate was and still is associated with muscle fatigue, but in the last decades it has been considered its significant modulating function of metabolism during exercise at cellular level and whole organism level. It has been shown that its effects might be similar to the effects of hypoxia and its oxidized form, pyruvate, has the cardioprotective effects. The aim of this study was to compare the expression of LDHA and LDHB isoforms between left and right ventricle in the cardioprotective scheme of adaptation to hypoxia. Another objective/goal was to determine the left ventricular response to I/R insult in the perfused heart model adapted to hypoxia compared with the normoxic controls on/at the expression level of both LDH isoforms. Our results showed differences in the LDHA expression in the left and right ventricle and an increased response of the left ventricle to I/R insult in rats adapted to hypoxia which is reflected at the expression level of both isoforms. Key words: heart,...

Background-Previously we found that smooth muscle cell (SMC)-specific knockout of miR-17 similar to 92 attenuates hypoxia-induced pulmonary hypertension. However, the mechanism underlying miR-17 similar to 92-mediated pulmonary artery SMC (PASMC) proliferation remains unclear. We sought to investigate whether miR-17 similar to 92 regulates hypoxia-inducible factor (HIF) activity and PASMC proliferation via prolyl hydroxylases (PHDs). Methods and Results-We show that hypoxic sm-17 similar to 92(-/-) mice have decreased hematocrit, red blood cell counts, and hemoglobin contents. The sm-17 similar to 92 (-/-) mouse lungs express decreased mRNA levels of HIF targets and increased levels of PHD2. miR-17 similar to 92 inhibitors suppress hypoxia-induced levels of HIF1 alpha, VEGF, Glut1, HK2, and PDK1 but not HIF2 alpha in vitro in PASMC. Overexpression of miR-17 in PASMC represses PHD2 expression, whereas miR-17/20a inhibitors induce PHD2 expression. The 3'-UTR of PHD2 contains a functional miR-17/20a seed sequence. Silencing of PHD2 induces HIF1a and PCNA protein levels, whereas overexpression of PHD2 decreases HIF1 alpha and cell proliferation. SMC-specific knockout of PHD2 enhances hypoxia-induced vascular remodeling and exacerbates established pulmonary hypertension in mice. PHD2 activator R59949 reverses vessel remodeling in existing hypertensive mice. PHDs are dysregulated in PASMC isolated from pulmonary arterial hypertension patients. Conclusions-Our results suggest that PHD2 is a direct target of miR-17/20a and that miR-17 similar to 92 contributes to PASMC proliferation and polycythemia by suppression of PHD2 and induction of HIF1 alpha.

Hypoxia inducible factors (HIFs) are transcription factors and the master regulators of oxygen-dependent gene expression in animals. The focus of this thesis is the distribution of HIF protein in tissues of the fish Fundulus heteroclitus and F. grandis, two widespread species that occur in naturally hypoxic waters. Polyclonal antibodies against HIF-1α, HIF-2α, and HIF-3α were tested on proteins made in vitro and on extracts made from several tissues of normoxic and hypoxic fish. Antibodies against HIF-1α and 3α bound specifically to full length protein made in vitro, and produced bands on western blots of nuclear extracts of near the expected molecular weights for these proteins. Hypoxic exposure did not markedly increase the intensity of these bands, and mass spectrometry failed to identify HIF-1α and 3α peptides in excised gel bands. Thus, further tests of antibody specificity are needed before the tissue distribution of HIF in these fish can be confidently assessed.

Abstract Oxygen is vital for human survival. To ensure its sufficient supply, the body has an intricate system, which involves the circulatory, respiratory and neuroendocrine systems. When oxygen is lacking, a state of hypoxia occurs, and adaptive changes in gene expression increase oxygen delivery to promote survival. The key regulator of the transcriptional hypoxia response is hypoxia-inducible factor (HIF) which targets over 1000 genes. The HIF prolyl 4-hydroxylases (HIF-P4Hs) govern the stability of HIF in an oxygen-dependent fashion. In our studies we investigated whether activation of the hypoxia response through inhibition of either of two distinct HIF-P4Hs, HIF-P4H-2 or P4H-TM would reduce atherosclerosis in mice. We found that inhibition of HIF-P4H-2 led to reductions in numbers of atherosclerotic plaques, and levels of serum cholesterol and inflammation in white adipose tissue and aortic plaques. In addition, HIF-P4H-2 deficient mice had elevated levels of modified LDL-targeting, atheroprotective circulating autoantibodies. The P4H-TM knockout mice also had reduced numbers of atherosclerotic plaques and increased levels of atheroprotective autoantibodies in their sera, but in contrast to the HIF-P4H-2 deficient mice, they also showed a reduction in serum triglyceride levels. To determine how hypoxia alters maternal glucose and lipid metabolism in pregnancy, we studied pregnant mice that were predisposed to a hypoxic condition (15% ambient O2). We found that they had enhanced glucose metabolism due to reduced insulin resistance and an increased flux of glucose to maternal tissues. The hypoxic dams also failed to gain weight and store adipose tissue in the anabolic phase to the same extent as normoxic control dams. These results implicate HIF-P4H inhibition as a novel therapeutic mechanism for atherosclerosis, and suggest that the small molecule HIF-P4H inhibitors currently in clinical trials for renal anemia may have further possible therapeutic applications. In addition, greater understanding of the changes in maternal metabolism that underly reduced fetal growth in hypoxic conditions, and the development of targeted interventions may allow the preservation of fetal growth in cases of maternal hypoxia. / Tiivistelmä Happi on ihmiselle elintärkeää. Tämän vuoksi meille on kehittynyt pitkälle jalostunut verenkierto-, hengitys- ja neuroendokriininen järjestelmä sekä sellaisten geenien ilmentymisen muutoksia, jotka joko lisäävät hapen kuljetusta tai auttavat selviytymään hypoksisissa oloissa, jotta taataan riittävä hapen saanti. Hapen puutteessa hypoksiavaste, jonka tärkein säätelijä on hypoksiassa indusoituva transkriptiotekijä (HIF), aktivoituu. HIF:lla on yli 1000 kohdegeeniä joiden kautta sen vaikutukset välittyvät. HIF-prolyyli-4-hydroksylaasit (HIF-P4H:t) säätelevät HIF:n stabiilisuutta hapesta riippuvaisesti. Tutkimuksessamme selvitimme, vähentääkö hypoksiavasteen aktivointi HIF-P4H-2:n tai P4H-TM:n inhibition kautta ateroskleroosia hiirillä. Tuloksena oli, että HIF-P4H-2:n inhibitio vähensi ateroskleroottisia plakkeja, seerumin kolesterolia ja inflammaatiota valkoisessa rasvakudoksessa sekä plakeissa. Lisäksi hiirillä, joilta puuttui HIF-P4H-2, oli lisääntynyt määrä ateroskleroosilta suojaavia muokattua LDL:ää sitovia autovasta-aineita seerumissa. P4H-TM-poistogeenisillä hiirillä todettiin vastaavasti vähemmän ateroskleroottisia plakkeja ja lisääntynyt määrä ateroskleroosilta suojaavia autovasta-aineita seerumissa. Poiketen HIF-P4H-2-puutteisista hiiristä, niillä oli matalammat seerumin triglyseridi-tasot. Tutkimme raskaina olevia hiiriä, jotka altistimme hypoksisille olosuhteille (15% O2), jotta pystyisimme määrittämään, kuinka hypoksia vaikuttaa äidin sokeri- ja rasva-aineenvaihduntaan. Hypoksiassa raskaana olevilla hiirillä todettiin tehostunut sokeriaineenvaihdunta, joka oli seurausta alentuneesta insuliiniresistenssistä sekä lisääntyneestä sokerin sisäänotosta äidin kudoksiin. Hypoksiassa eivät raskaana olevien hiirten paino eivätkä rasvavarastot lisääntyneet samassa suhteessa normoksiassa raskaana olevien hiirten kanssa. Nämä tulokset tarjoavat uusia mahdollisuuksia HIF-P4H-inhibition käyttämiseen terapeuttisena vaihtoehtona ateroskleroosin hoidossa ja ehkäisemisessä. Kliinisissä kokeissa munuaisperäisen anemian hoidossa olevat HIF-P4H-estäjät voisivat näin ollen saada lisää indikaatioita. Lisäksi korkean ilmanalan aiheuttaman pienipainoisuuden takana olevien aineenvaihdunnan muutoksien ymmärtäminen voi mahdollistaa sikiön kasvun turvaamisen spesifein interventioin.

A galectina-3 (gal-3) pertence a uma família de proteínas com domínios de ligação a beta-galactosídeos e está relacionada com diversos aspectos tumorais, como proliferação e adesão celular, angiogênese e proteção contra morte celular. Estudos mostram sua relação com o fenômeno da hipóxia, característica de diversos tumores sólidos que apresentam altas taxas de proliferação celular. A adaptação à hipóxia é mediada principalmente pelo Fator Induzido por Hipóxia (HIF-1), a qual atua na indução de diversos genes de sobrevivência em ambientes com baixas concentrações de oxigênio. Além de HIF, outros fatores são importantes nesse processo, como NF-kB, por exemplo, sendo um fator de transcrição responsivo a diversos estresses celulares, entre eles, a hipóxia. Alguns modelos tumorais apresentam-se ideais para o estudo dos efeitos da hipóxia no microambiente tumoral, como os glioblastomas. Estes são tumores do sistema nervoso central com altas taxas de letalidade, são refratários aos principais métodos de tratamento por sua plasticidade, crescimento infiltrativo e heterogeneidade. Histologicamente, estes tumores apresentam atipia nuclear, altas taxas de mitose e áreas de pseudopaliçada. Postula-se que estas áreas sejam compostas por células migrantes de ambientes necróticos, os quais são também hipóxicos devido a sua distância de vasos sanguíneos e é demonstrado que estas células expressam tanto HIF-1alfa quanto gal-3. Ensaios in vitro realizados por nosso grupo demonstraram que a gal-3 é positivamente regulada pela hipóxia em uma linhagem de glioma híbrido, NG97ht, além de demonstrar que esta proteína é um fator chave na proteção destas células contra a morte celular induzida pela privação de oxigênio e nutrientes, mimetizando condições necróticas de pseudopaliçada in vivo, destacando-se as habilidades antiapoptóticas desta proteína. Embora uma de suas possíveis funções tenha sido elucidada, os mecanismos de atuação e de indução da gal-3 ainda são obscuros. Deste modo, este projeto visa explorar os papéis pró-tumorais da gal-3, podendo torná-la um possível alvo em terapias anti-neoplásicas, entendendo melhor seus mecanismos de proteção contra a morte celular e controle de expressão em ambientes hipóxicos, além de estudar suas possíveis funções in vivo no desenvolvimento de tumores, e também estendendo seus estudos para outras linhagens de glioblastoma. Nossos resultados demonstraram que a gal-3 está co-localizada com mitocôndrias nestas linhagens de glioma, podendo sofrer alterações pós-traducionais em hipóxia, como a fosforilação e que houve acúmulo de HIF-1alfa nuclear nestas células em hipóxia. Vimos também que a gal-3 na linhagem NG97ht apresentou-se proveniente de dois alelos diferentes e que fatores intermediários deveriam ser expressos previamente pela célula antes da indução de gal-3 em hipóxia. Também demonstramos que houve dependência de NF-kB na indução transcricional de gal-3 nestas condições. Estes experimentos também demonstraram que a exposição de células à hipóxia e privação de nutrientes é capaz de induzir tanto espécies reativas de oxigênio como o aumento da autofagia nestas células, fatores importantes na indução da morte celular, além de demonstrar que na linhagem NG97ht a indução da morte nestas condições ocorreu por necrose, sem apresentar apoptose celular. Expandimos esta teoria da participação da gal-3 como molécula protetora contra a morte em hipóxia e privação de nutrientes para outra linhagem de glioma humano, a T98G. E finalmente, demonstramos que a diminuição da expressão de gal-3 em células tumorais da linhagem U87MG levou a diminuição das taxas de estabelecimento e crescimento tumoral in vivo / Galectin-3 (gal-3) belongs to a family of proteins with beta-galactoside binding domains and is related to various tumoral aspects, such as cell proliferation and adhesion, angiogenesis and protection against cell death. Studies show its relationship with the hypoxia phenomenon, a characteristic of many solid tumors that have high cell proliferation rates. The adaptation to hypoxia is mainly mediated by Hypoxia Induced Factor (HIF-1), which acts in the induction of several survival genes in environments with low oxygen concentrations. In addition to HIF, other factors are important in this process, such as NF-kB, for example, which is a transcription factor responsive to various cellular stresses, including hypoxia. Some tumor models are ideal for studying the effects of hypoxia in the tumor microenvironment, e.g. glioblastomas. These central nervous system tumors with high mortality rates are refractory to the main treatment methods due to their plasticity, heterogeneity and infiltrative growth. Histologically, these tumors exhibit nuclear atypia, high mitotic rates and pseudopalisading areas. It is postulated that these areas are composed of migrating cells out of necrotic microenvironments, which are also hypoxic due to their distance from the blood vessels and it is shown that these cells express both HIF-1alfa and gal-3. In vitro assays performed by our group demonstrated that gal-3 is positively regulated by hypoxia in a hybrid glioma cell line, NG97ht, and demonstrated that this protein is a key factor in protecting these cells against cell death induced by oxygen and nutrient deprivation conditions mimicking necrotic pseudopalisading areas in vivo, highlighting the pro-survival abilities of this protein. Although one of its possible functions has been elucidated, gal-3 mechanisms of action and induction are still unclear. Thus, this project aims to explore the gal-3 pro-tumoral effects, which may make it a possible target for anti-neoplastic therapies, better understanding the mechanisms of protection against cell death and expression in hypoxic environments, and also study its possible functions in vivo, extending these studies to other glioma cell lines. Our results demonstrated that gal-3 is located within the mitochondria in these glioma cell lines and may undergo posttranslational modifications in hypoxia, such as phosphorylation and that there is accumulation of nuclear HIF-1alfa in these cells under hypoxia. We have also seen that gal-3 in the NG97ht cell line presents two different alleles and that intermediate factors must be expressed previously by the cell before gal-3 induction in hypoxia. We also demonstrated that there is dependence on the NF-kB transcriptional factor for the gal-3 induction under these conditions. These experiments also demonstrated that exposure of cells to hypoxia and nutrient deprivation is capable of inducing reactive oxygen species and increased autophagy in these cells, which are important factors in the induction of cell death. In addition, we demonstrated that the induction of the NG97ht cell death in these conditions is due to necrosis. We expanded this theory of the participation of gal-3 as a protective molecule against cell death in hypoxia and nutrient deprivation to another human glioma cell line, T98G. And finally, we demonstrated that decreased expression of gal-3 in the U87MG glioma cell line leads to lower tumor establishment rates and decreased growth in vivo