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.

Angiogenesis is known to be induced and maintained in tumors by the constant expression of the hypoxia inducible factor 1 alpha (HIF-1α) and human vascular endothelial growth factor (VEGF). In fact, tumor recurrence, aggressive metastatic legions and patient mortality rates are known to be positively correlated with overexpression of these two proteins. The HIF-1α and VEGF promoters contain a polypurine/polypyrimidine (pPu/pPy) tract, which are known to play critical roles in their transcriptional regulation, and are structurally dynamic where they can undergo a conformational transition between B-DNA, single stranded DNA and atypical secondary DNA structures such as G-quadruplexes and i-motifs. We hypothesize that the i-motif and G-quadruplex structures can form within the pPu/pPy tracts of the HIF-1α and VEGF proximal promoters, which play important roles in the transcriptional regulation of these genes by acting as scaffolds for alternative transcription factor binding sites. The purpose of this dissertation was to elucidate the transcriptional regulation of the HIF-1α and VEGF genes through the atypical DNA structures that form within the pPu/pPy tracts of their proximal promoters. We investigated the interaction of the C-rich and guanine-rich (G-rich) strands of both of these tracts with transcription factors heterogeneous nuclear ribonucleoprotein (hnRNP) K and nucleolin, respectively, both in vitro and in vivo and their potential role in the transcriptional control of HIF-1α and VEGF. In this dissertation, we demonstrate that both nucleolin and hnRNP K bind selectively to the G- and C-rich sequences, respectively, in the pPu/pPy tract of the HIF-1α and VEGF promoters. Specifically, the small interfering RNA-mediated silencing of either nucleolin or hnRNP K resulted in the down-regulation of basal VEGF gene, and the opposite effect was seen when the transcription factors were overexpressed, suggesting that they act as activators of VEGF transcription. Taken together, the identification of transcription factors that can recognize and bind to atypical DNA structures within pPu/pPy tracts will provide new insight into mechanisms of transcriptional regulation of the HIF-1α and VEGF gene.

Cancer develops in many organs and tissues in the body through genetic and environmental modifications to acquire the hallmarks of cancer. The hallmarks of cancer allow the cells to become malignant and progress to a tumorigenic state. It has previously been shown in various carcinomas that HIF-2a, a key component in hypoxia adaptation, has a role in autonomous growth, the first hallmark of cancer. Ovarian cancer is the most lethal of the gynecological malignancies and accounts for 3% of new cases in women annually but is the fifth most common cause of death due to cancer. Here, it is shown in two ovarian carcinoma cell lines that HIF-2a is involved in in vitro and in vivo growth. It is also shown that the effect of HIF-2a is due to its role in autonomous growth and not vascularization with the use of in vitro spheroids. From recent findings in the laboratory the oxygen-stimulated translation initiation complex was discovered and HIF-2a is one of its components. In the absence of HIF-2a there is a downregulation in translation in hypoxia in ovarian carcinoma. This is also seen in a HIF-2a translational target, IGF1R and its downstream signaling pathway, which may be involved in autonomous growth as well as other hallmarks of cancer. Taken together, the data in this thesis presents the importance of HIF-2a in autonomous growth and cancer progression in ovarian carcinoma, as well as verifying its role in translation.

The complex pathobiologic mechanisms of emphysema are not fully understood, leaving this deadly disease without effective pharmacotherapy for a cure. This project hypothesized that the sulfated dehydropolymer of caffeic acid (CDSO3) exhibits Fe2+ chelation-based hypoxia inducible factor-1a (HIF-1a) up-regulatory protective activities against in vitro emphysematous cell death and for in vivo reversal of emphysema induced with SU5416, a vascular endothelial growth factor blocker. Using in vitro chromogenic competitive inhibition assays, CDSO3 was shown to chelate Fe2+ (IC50 of 23 µM), but not Fe3+ ions. The trypan blue exclusion and lactate dehydrogenase assays were then employed to examine the cytoprotective activities of CDSO3 against inflammatory, oxidative, elastolytic, and apoptotic cell death using alveolar macrophages, epithelial and endothelial cells. CDSO3 at 10 µM produced significant protective activities against these emphysematous cell deaths by 50-154 %. These protective effects were opposed by the addition of the HIF-1a inhibitors, CAY10585 and echinomycin, and excess Fe2+, but not Fe3+, ions. Emphysema was then induced in rats following a subcutaneous injection of SU5416 at 20 mg/kg, after which CDSO3 at 60 µg/kg was administered to the lungs 3 times/week for two weeks. Treadmill exercise endurance (EE) was measured to assess the functional impairment, while lung tissues were removed for morphological assessments of alveolar airspace enlargement (MLI) and destruction (DI), as well as to measure protein levels using Western blot. SU5416 significantly impaired EE, MLI, and DI by 81 %, 47 %, and 5-fold, compared to the healthy animals, and these were significantly reversed by CDSO3 by 66, 74, and 87 %. CDSO3 treatment did not change the lung cytoplasmic expression of histone deacetylase 2 (HDAC2), HIF-1a, or a pro-apoptotic marker, BAX. However, induction with SU5416 significantly reduced VEGF expression by 52 % and increased cleaved caspase-3 expression by 1.5-fold, compared to the healthy animals, while CDSO3 normalized the expressions of both proteins in these emphysematous animals. However, when CDSO3 was pre-mixed with excess Fe2+, the reversal activities of CDSO3 were diminished. In conclusion, this study has demonstrated the Fe2+ chelation-based HIF-1a up-regulatory dependent in vitro and in vivo lung repairing efficacies for CDSO3 in emphysema.

PARP-1 has been identified as a major player in apoptotic pathways. Its excessive activation causes mitochondrial dysfunction, permeability, and AIF mitochondrion-to-nucleus translocation. It has been suggested that PARP-1 interacts indirectly with Bnip3, a mitochondrial pro-apoptotic factor. However, the mechanistic linkage is still not well understood. Our lab has shown that cytosolic/nuclear NAD+ depletion is a hallmark for PARP-1 over activation and inhibition of sirtuin activity. Specifically in my project, we think that PARP-1 induced- NAD+ depletion and sirtuin inhibition causes hyperacetylation of the α subunit of the transcription factor HIF-1 allowing increased HIF-1 binding to Bnip3 upstream promoter, and increased Bnip3 expression. Indeed, our PARP-1 Knock out neurons, MNNG and PJ34 treatment, chromatin immunoprecipitation, and HIF-1α loss of function studies strongly confirmed the necessity of HIF-1 to increase Bnip3 expression in hypoxia. Overall, our research suggests a role for HIF-1 in increasing PARP-1 dependent Bnip3 expression in hypoxic models. / May 2016

Hypoxia (low tissue oxygenation) is an important signalling cue for many cell types. The study of its effects has direct relevance to surgery since hypoxic gradients are generated with every cut. On a cellular level, changes in molecular oxygen are sensed by the Hypoxia-Inducible Factors (HIFs). The HIFs are a family of transcription factors that are master regulators of over 100 genes and can effect changes in multiple cellular processes including migration, survival and differentiation. The broad nature of the response to hypoxia means that study of the HIF system is also important in cancer; where many tumour cells have found ways of subverting the HIF response to ensure their continued growth and survival. This thesis explores the role of hypoxia and the HIF system in the regulation of migration, survival and differentiation in both cancer and stem cells. The first experimental chapter examines the role of hypoxia and the HIF system in the regulation of migration and three-dimensional organisation in several cancer cell lines. Using biochemical and functional assays, the HIF system is shown to exert a pleiotropic effect across a panel of cancer cell lines. In particular, HIF 1α is shown to activate proliferation in a prostate cancer cell line in findings that may be useful to inform future clinical strategies for the management of this disease. In the second experimental chapter, the first epigenetic mechanism involving histone modification for the specific regulation of HIF 2α expression is characterised. Here the family with sequence similarity 60, member A (Fam60a) protein is shown to repress expression of the HIF 2α gene through its association with the class 1 Sin3-HDAC co-repressor complex, achieving specificity by co-operation with the SP1 transcription factor. This novel mechanism is demonstrated to be important in the regulation of the basal expression of HIF 2α. Modification of HIF 2α expression through this mechanism is shown to alter cell migration, three dimensional organisation and angiogenesis in vitro. The clinical importance of these findings is demonstrated in a series of 45 patients suffering from colorectal cancer of known stage. In this cohort, the reciprocal relationship between Fam60a and HIF 2α is maintained, and both are identified as potential novel biomarkers for the development of this disease. In the final experimental chapter, the role of hypoxia in the regulation of differentiation is explored. These effects are documented in mesenchymal progenitors primarily derived from human fat. Here, hypoxia is shown to regulate differentiation in a context-dependent manner, promoting osteogenic and retarding adipose and neural differentiation in-vitro. The roles of Fam60a and HIF 2α are explored in this system. These data may be useful in optimising future surgical engraftment of these cells for regenerative purposes.

Changes to the metabolism of the cardiomyocyte are driven by complex signaling pathways in order to adjust to stress. For instance, HIF-1α is classically known to upregulate glycolytic metabolism to compensate for oxygen deficiency. Other important effects upon glucose metabolism, which we investigate here more extensively, were also observed. Hearts derived from mice with the cardiac-restricted expression of a stabilized form of HIF-1α are remarkably ischemia stress-tolerant. Here, stable isotope-resolved metabolomic analyses were utilized to investigate glucose cardiometabolism remodeling by HIF-1αduring ischemia. We found that 13C-lactate accumulation was significantly elevated in HIF-1α expressing hearts while paradoxically glycogen was maintained to a remarkable extent during an ischemic time course. These findings suggested an unexpected source of glucose in HIF-1α hearts during global ischemia. Accordingly, the presence of gluconeogenesis in hearts was evaluated. Indeed, gluconeogenic intermediates (i.e. m+3) including glucose-6-phosphate [m+3], fructose-6-phosphate [m+3], and fructose 1,6-bisphosphate [m+3] were observed at significantly elevated levels in the ischemic HIF-1α heart. Collectively, these data establish the surprising finding that HIF-1α supports active gluconeogenesis in the heart during ischemia. As less is known regarding the effects of CTRP3 we first tested whether CTRP3 overexpression would protect the ischemic heart. Our data indicate that CTRP3 failed to confer ischemic tolerance in heart ex vivo. However,we were able to show that CTRP3 protected the liver from lipid-induced stress and prevented hepatic lipid accumulation. To further investigate the mechanisms of hepatic protective effect mediated by CTRP3, we identified the receptor and established that CTRP3 increases oxygen consumption in response to lipid overloaded. Lysosomal-associated membrane protein 1 (LAMP-1), In summary, these data indicate that targeted metabolic rearrangements within cardiomyocyte/hepatocyte holds promise for the alleviation of common pathological conditions.

La polyglobulie (PG) a un arrière plan clinique et physiopathologique varié, et dont la connaissance a beaucoup évolué depuis 5 ans. Elle a pour caractéristique nécessaire et commune une augmentation du volume globulaire total qui peut résulter de mécanismes moléculaires différents. L'érythrocytose est une forme de la PG ou` seule la lignée érythroïde semble être augmentée. Dans ce contexte, nous nous sommes intéressés à la mutation JAK2 (Val617Phe) et aux gènes EPOR, VHL, PHD2 chez des patients présentant une érythrocytose d'origine inconnue (98 familles). Nous avons trouvé 3 nouvelles mutations délétionnelles et d'autres mutations faux-sens et silencieuses sur EPOR. Les cellules FDCP-1 et 32D transfectées, qui expriment les récepteurs tronqués ont montré une hypersensibilité à l'Epo à concentration basse. Les analyses Epo-dose-réponse pour une mutation faux-sens étaient semblables à celles du type sauvage. La mutation JAK2(Val617Phe) était présente avec une faible fréquence dans la série de patients étudiés. La recherche des mutations dans le gène PHD2 nous a permis de trouver 3 nouvelles mutations frame-shift et non-sens, les seules connues à ce jour: Elles entraînent la perte d'une partie ou de l'ensemble du site catalytique de PHD2 dans l'hypothèse où elles seraient synthétisées. Ainsi, ces mutations et les deux autres mutations faux-sens décrites démontrent l'importance de PHD2 dans la régulation de la voie de HIF-α, et qu'une perturbation de cette régulation pourrait conduire à une PG. La majorité des patients reste sans défaut moléculaire identifié, et des recherches complémentaires sont nécessaires. Nous avons également étudié deux Hbs augmentant l'affinité pour l'oxygène: les Hbs Nantes et La Coruña. Enfin, nous avons cherché des mutations dans les 3 exons de DPGM chez 4 patients avec un taux de 2,3-DPG bas. Une seule mutation a été trouvée dans la région 5`, dans l'exon 1 non traduit, elle nécessite des études complémentaires.

Cancer develops in many organs and tissues in the body through genetic and environmental modifications to acquire the hallmarks of cancer. The hallmarks of cancer allow the cells to become malignant and progress to a tumorigenic state. It has previously been shown in various carcinomas that HIF-2a, a key component in hypoxia adaptation, has a role in autonomous growth, the first hallmark of cancer. Ovarian cancer is the most lethal of the gynecological malignancies and accounts for 3% of new cases in women annually but is the fifth most common cause of death due to cancer. Here, it is shown in two ovarian carcinoma cell lines that HIF-2a is involved in in vitro and in vivo growth. It is also shown that the effect of HIF-2a is due to its role in autonomous growth and not vascularization with the use of in vitro spheroids. From recent findings in the laboratory the oxygen-stimulated translation initiation complex was discovered and HIF-2a is one of its components. In the absence of HIF-2a there is a downregulation in translation in hypoxia in ovarian carcinoma. This is also seen in a HIF-2a translational target, IGF1R and its downstream signaling pathway, which may be involved in autonomous growth as well as other hallmarks of cancer. Taken together, the data in this thesis presents the importance of HIF-2a in autonomous growth and cancer progression in ovarian carcinoma, as well as verifying its role in translation.

Marking proteins for degradation by the proteasome is a classical function of ubiquitination. This process of covalent attachment of a chain of ubiquitin molecules to target proteins is governed by the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2) and the ubiquitin ligase (E3). The von Hippel-Lindau (VHL) tumour suppressor protein forms an E3 ubiquitin ligase, ECV (Elongins BC/Cul2/VHL), which targets the alpha subunit of hypoxia-inducible factor (HIF) for ubiquitin-mediated destruction under normal oxygen tension. Tumour hypoxia promotes accumulation of HIFalpha, whose expression is associated with cancer progression, poor prognosis and resistance to conventional therapies, thus establishing HIF as a therapeutic target. Notably, VHL is functionally inactivated in VHL disease, a hereditary cancer syndrome characterized by the formation of tumours in multiple organs, as well as in the majority of sporadic clear-cell renal cell carcinomas (CCRCC) and haemangioblastomas. Recently, certain VHL mutations have been shown to cause the congenital disorder Chuvash polycythemia. Work contained in this thesis describes the temporally coordinated activation of the ECV, whereby oxygen-dependent recognition of HIFalpha by VHL triggers Cul2 modification by the ubiquitin-like molecule NEDD8, which enhances ECV ubiquitin ligase activity by recruiting the E2. In addition, the feasibility of ‘bio-tailored’ enzymes in the treatment of cancer is introduced by creating a bioengineered VHL capable of targeting HIFalpha for degradation irrespective of oxygen tension, which leads to the dramatic inhibition of CCRCC tumour growth and angiogenesis in a xenograft model. Furthermore, a ubiquitin ligase composed of two F-box proteins, VHL and suppressor of cytokine signalling 1 (SOCS1), was identified and shown to be paramount for the negative regulation of erythropoiesis by targeting phosphorylated Janus kinase 2 (JAK2) for ubiquitin-mediated destruction. The malfunction of this ubiquitin ligase explains the excessive erythrocytosis observed in Chuvash polycythemia patients and reveals a novel genetic link between the seemingly distinct genes VHL and JAK2 in the development of polycythemia.