Abstract Oxygen is essential for aerobic organisms, as shortage of oxygen (hypoxia) can induce cellular dysfunctions and even cell death, leading to tissue damage and decreased viability of the organism. Oxygen homeostasis is regulated delicately by several mechanisms, the major one being the hypoxia-inducible factor (HIF) pathway that is evolutionarily conserved. HIFα subunits are regulated in an oxygen-dependent manner via three HIF prolyl 4-hydroxylases (HIF-P4Hs). In the presence of oxygen HIF-P4Hs modify HIFα, which leads to its degradation, whereas in hypoxia the HIF-P4H enzymes cannot function and HIFα is stabilized. HIF regulates more than 300 genes that enhance oxygen delivery from the lungs to tissues and reduce oxygen consumption in tissues, such as those for erythropoietin and vascular endothelial growth factor. When a tissue suffers from hypoxia caused by a circulatory restriction, the situation is called ischemia. In this study we used a genetically modified HIF-P4H-2 hypomorph mouse line that expresses 8% of the wild-type Hif-p4h-2 mRNA in the heart and 19% in skeletal muscle, and has HIFα stabilization in both tissues. We showed that chronic HIF-P4H-2 deficiency leads to protection against acute ischemic injury both in the heart and in skeletal muscle. The protection was mainly due to enlarged capillaries and better perfusion in both tissues. Hypoxia is known to decrease body weight. The observation of the HIF-P4H-2 deficient mice being leaner than their wild-type littermates led us to study their body constitution, metabolism and adipose tissue in detail. We discovered that chronic HIF-P4H-2 deficiency protects against obesity and several metabolic dysfunctions including diabetes and metabolic syndrome. These beneficial outcomes were mimicked when a pharmacological pan-HIF-P4H inhibitor was administered to wild-type mice. In these studies we showed that pharmacological HIF-P4H-2 inhibition may provide a novel treatment for diseases such as acute myocardial infarction, peripheral artery disease and metabolic disorders. / Tiivistelmä Happi on edellytys aerobisen eliön, kuten ihmisen, elämälle; hapen niukkuus (hypoksia) voi johtaa monenlaisiin solun toimintahäiriöihin, jotka voivat edelleen aiheuttaa solun kuoleman, kyseisen kudoksen vaurion, ja lopulta eliön elinkyvyn heikkenemisen. Happitasapainoa säädellään monilla menetelmillä, joista merkittävin on hypoksiassa indusoituvasta tekijästä (HIF) riippuvainen reitti, joka on evoluutiossa säilynyt. HIFα alayksiköitä säätelee hapesta riippuvaisesti kolme HIF prolyyli 4-hydroksylaasia (HIF-P4Ht). Hapen läsnä ollessa HIF-P4H on aktiivinen ja johtaa HIFα:n hajottamiseen, kun taas hypoksiassa HIF-P4H entsyymit eivät voi toimia ja siten HIFα stabiloituu. HIF säätelee yli 300 geeniä, jotka edistävät hapen kuljetusta ja pääsyä keuhkoista kudoksiin sekä vähentävät hapenkulutusta. Näitä geenejä ovat mm. erytropoietiini sekä vaskulaarinen endoteelikasvutekijä. Kudoksen heikentyneestä verenkierrosta johtuvaa hapenpuutetta kutsutaan iskemiaksi. Tässä tutkimuksessa käytimme geneettisesti muunneltua HIF-P4H-2 hypomorfi-hiirikantaa, joka tuottaa Hif-p4h-2 lähetti-RNA:ta sydämessä vain 8 % ja luurankolihaksessa 19 % villityypin määrästä, ja jolla on HIFα stabiloituneena molemmissa kudoksissa. Osoitimme, että krooninen HIF-P4H-2:n puute suojaa sekä sydäntä että luurankolihasta akuutissa iskemiassa. Vaikutus johtui pääasiassa suuremmista kapillaareista ja paremmasta perfuusiosta molemmissa kudoksissa. Aikaisempien tutkimusten perusteella tiedetään, että hypoksia alentaa painoa. Huomio siitä, että HIF-P4H-2 puutteiset hiiret ovat hoikempia kuin villityypin sisaruksensa, johti meidät tutkimaan hiirten kehon koostumusta, aineenvaihduntaa ja rasvakudosta tarkemmin. Tutkimuksissamme selvisi, että krooninen HIF-P4H-2:n puute suojaa lihavuudelta ja monelta aineenvaihdunnan häiriöltä kuten sokeritaudilta ja metaboliselta oireyhtymältä. Nämä edulliset vaikutukset toistuivat, kun annoimme villityypin hiirille pan-HIF-P4H inhibiittoria. Kaiken kaikkiaan, näissä tutkimuksissa osoitimme, että lääkkeellinen HIF-P4H-2:n estäminen voi tarjota uuden keinon sydäninfarktin, luurankolihasiskemian ja aineenvaihdunnan häiriöiden hoitoon.

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AMP-activated protein kinase (AMPK) is known as a pivotal cellular energy sensor, mediating the adaptation to low energy levels by deactivating anabolic processes and activating catabolic processes in order to restore the cellular ATP supply when the cellular AMP/ATP ratio is increased. Besides this well-known role, it has also been shown to exert protective effects under hypoxia. While an insufficient supply with oxygen might easily deplete cellular energy levels, i.e., ATP concentration, manifold other mechanisms have been suggested and are heavily disputed regarding the activation of AMPK under hypoxia independently from cellular AMP concentrations. However, an activation of AMPK preceding energy depletion could induce a timely adaptation reaction preventing more serious damage. A connection between AMPK and the master regulator of hypoxic adaptation via gene transcription, hypoxia-inducible factor (HIF), has also been taken into account, orchestrating their concerted protective action. This review will summarize the current knowledge on mechanisms of AMPK activation under hypoxia and its interrelationship with HIF.

Οι όγκοι εγκεφάλου αποτελούν μια ομάδα όγκων με ιδιαίτερα χαρακτηριστικά. Η ογκογένεση έχει συσχετισθεί σε αρκετούς όγκους, ανάμεσά τους και ορισμένοι όγκοι εγκεφάλου, με την πρωτείνη θερμικού σοκ 90 (Hsp90). Σε πρόσφατες μελέτες, έχει αποδειχθεί η η συμμετοχή της Hsp90 στην αποδόμηση της ογκοκατασταλτικής πρωτείνης pVHL, η οποία είναι απαραίτητητη για την αποδόμηση του μεταγραφικού παράγοντα που ενεργοποιείται στην υποξία (HIF-1α). Επιπλέον, έχει βρεθεί πως οι ανταγωνιστές της Hsp90 μειώνουν τα επίπεδα έκφρασης του VEGFR-3. Στόχος της παρούσας μελέτης ήταν η εκτίμηση των επιπέδων έκφρασης των μορίων Hsp90, pVHL, HIF-1α και VEGFR-3 στους όγκους εγκεφάλου και οι πιθανές συσχετίσεις μεταξύ τους. Εξετάσθηκαν συνολικά 89 óγκοι εγκεφάλου με την ανοσοϊστοχημική μέθοδο. Στους αστροκυτταρικούς όγκους, η Ηsp90 φαίνεται πως οδηγεί στη σταθεροποίηση του HIF-1α μέσω της σύνδεσής της με τη pVHL. Στα μυελοβλαστώματα από την άλλη, ο HIF-1α ρυθμίζεται από τη Hsp90 με τρόπο ανεξάρτητο από τη pVHL. Βρέθηκε συσχέτιση της έκφρασης του VEGFR-3 με τη Hsp90 αλλά και τον HIF-1α σε ομάδα όγκων του εγκεφάλου, η βιολογική συμπεριφορά των οποίων θα πρέπει να διερευνηθεί. / Brain tumors constitute a special group of tumors. In many tumors, including brain tumors, tumorigenesis has been associated with heat shock protein 90 (Hsp90). ). Recent studies have demonstrated that Hsp90 is essential for the degradation of tumor suppressor protein, pVHL, which is essential for the degradation of Hypoxia Induced Factor 1α (HIF-1α). In addition,it has been proved that Hsp90 antagonists, reduce the expression VEGFR-3. the aim of the current study was to estimate the levels of expression of Hsp90, pVHL, HIF-1α and VEGFR-3 in brain tumors and the possible correlations among them.89 human brain tumors were studied immunohistochemically. In astrocytic tumors, Hsp90 seems to stabilize HIF-1α, through binding to pVHL. On the other hand, in medulloblastomas, HIF-1α seems to be regulated by Hsp90, in a way that seems to be independent from pVHL. We found a correlation between the expression of VEGFR-3 and Hsp90 and HIF-1α in a group of brain tumors, the biological behavior of which must be studied.

Traumatic brain injury (TBI) is becoming a national epidemic, as it accounts for 1.5 million cases each year. This disorder affects primarily the young population and elderly. Currently, there is no treatment for TBI, which means that ~2% of the U.S. population is currently living with prolonged neurological damage and dysfunction. Recently, there have been many studies showing that TBI negatively impacts mitochondrial function. It has been proposed that in order to save the cell from destruction mitochondrial function must be preserved. The ketogenic diet, originally designed to mimic fasting physiology, is effective in treating epilepsy. Therefore, we have used fasting as a post injury treatment and attempted to elucidate its underlying mechanism. 24 hours of fasting after a moderate TBI increased tissue sparing, cognitive recovery, improved mitochondrial function, and decreased mitochondrial biomarkers of injury. Fasting results in hypoglycemia, the production of ketones, and the upregulation of free fatty acids (FFA). As such, we investigated the neuroprotective effect of hypoglycemia in the absence of fasting through insulin administration. Insulin administration was not neuroprotective and increased mortality in some treatment groups. However, ketone administration resulted in increased tissue sparing. Also, reduced reactive oxygen species (ROS) production, increased the efficiency of NADH utilization, and increased respiratory function. FFAs and uncoupling proteins (UCP) have been implicated in an endogenously regulated anti-ROS mechanism. FFAs of various chain lengths and saturation were screened for their ability to activate UCP mediated mitochondrial respiration and attenuate ROS production. We also measured FFA levels in serum, brain, and CSF after a 24 hour fast. We also used UCP2 transgenic overexpressing and knockout mice in our CCI injury model, which showed UCP2 overexpression increased tissue sparing, however UCP2 deficient mice did not show a decrease in tissue sparing, compared with their wild type littermates. Together our results indicate that post injury initiated fasting is neuroprotective and that this treatment is able to preserve mitochondrial function. Our work also indicates ketones and UCPs may be working together to preserve mitochondrial and cellular function in a concerted mechanism, and that this cooperative system is the underlying mechanism of fasting induced neuroprotection.

This research programme investigated the expression of prolyl hydroxylase (PHD) proteins in rodent tissues. The importance of PHD enzymes lies in their ability to render oxygen sensitivity to Hypoxia inducible factor (HIF), the principal mediator of intracellular oxygen homeostasis. The first part of this study focused on developing and validating anti-sera capable of detecting PHD proteins in rodent tissues. With these reagents, it was possible to assess the relative expression of each PHD protein in a number of different rat tissues. PHD2 was the most abundant isoform in all tissues studied. In contrast, an abundance of PHD1 was observed only in testis and skeletal muscle. A number of different tissue species of PHD3 were identified and their abundance was found to vary between different tissues. These observations provide further evidence of the principal role of PHD2 in regulating HIF in vivo, but also point towards additional roles for PHD1 and PHD3 in selected tissues. They highlight the potential for there being a complex interplay between different PHD enzymes which could, in the future, prove potential targets for therapeutic manipulation. This study also provides additional insights into the mechanisms underlying the phenotypes observed in PHD deletional mouse models which appear, in many cases, to be directly related to the abundance of a given PHD isoform. The emerging role of PHD3 as a promoter of sympathetic lineage apoptosis prompted further study of PHD3 expression in rat neuronal tissues. An abundance of PHD3 was demonstrated throughout the rat sympathetic nervous system, a finding which appeared at odds with its known role as a promoter of neuronal apoptosis and resulted in a series of collaborative studies which demonstrated a sympatho-adrenal phenotype in wild type compared to PHD3-/- mice. Further collaborative studies utilising wild type mice and those deleted of specific PHD isoforms, were carried out to assess the significance of the abundance of PHD3 and PHD1 noted here in rat hippocampus and testis respectively. While neither study demonstrated statistically significant phenotypes, these observations remain of interest and areas for future research.

In our studies we found that stabilized expression of HIF-1α in heart led to better recovery of function and less tissue death after 30 minutes of global ischemia, via mechanisms that preserve the mitochondrial polarization. Our group previously showed that HIF-1α conferred ischemic tolerance by allowing cardiomyocytes to use fumarate as an alternative terminal electron acceptor to sustain anaerobic mitochondrial polarization. The source of fumarate was identified as the purine nucleotide cycle (PNC). Here we discovered that HIF-1α upregulates AMP deaminase 2 (AMPD2), the entry point to the PNC. The combination of glycolysis and the PNC may protect the heart's nucleotide resources. We subsequently examined the effects that HIF-1α exerts on nucleotide metabolism in the ischemic heart. We found that HIF-1α expression reduces adenosine accumulation in the ischemic heart. As ATP is depleted during ischemia, AMP accumulates. Our results suggest that AMP metabolism is shunted towards AMPD2 rather than the adenosine producing 5'-nucleotidase pathway. Subsequently, we treated hearts with the PNC inhibitor hadacidin followed by 30 minutes of global ischemia. Inclusion of hadacidin reduced ATP and adenylate energy charge in the hearts. These findings allow us to propose that activity of the PNC prevents the F0F1 ATP synthase from consuming glycolytic ATP in order to maintain mitochondrial polarization during ischemia. Thus, the PNC provides ATP sparing effects and preserves the energy charge in the ischemic heart. The fact that ATP and adenylate energy charge is better preserved during the initial 20 minutes of ischemia in HIF-1α expressing hearts is supportive of our observation that HIF-1α upregulates the PNC. HIF-1α also upregulates adenosine deaminase, which degrades adenosine. The limitation of adenosine accumulation may help HIF-1α expressing hearts avoid toxicity due to chronic adenosine exposure. Finally, we found that HIF-1α induces the expression of the nucleotide salvage enzyme hypoxanthine phosphoribosyl transferase (HPRT). Upon reperfusion HPRT serves to reincorporate the nucleotide degradation product, hypoxanthine, into the adenylate pool and may prevent the production of reactive oxygen species. Collectively, HIF-1α robustly protects the heart from ischemic stress and it upregulates several pathways whose cardioprotective role may extend beyond the remodeling of nucleotide metabolism.

The ability of cells to sense oxygen is a highly evolved process that facilitates adaptations to the local oxygen environment and is critical to energy homeostasis. In vertebrates, this process is largely controlled by three intracellular prolyl-4-hydroxylases (PHD 1-3). These related enzymes share the ability to hydroxylate the hypoxia-inducible transcription factor (HIF), and therefore control the transcription of genes involved in metabolism and vascular recruitment. However, it is becoming increasingly apparent that proline-4-hydroxylation controls much more than HIF signaling, with PHD3 emerging as the most unique and functionally diverse of the PHD isoforms. In fact, PHD3-mediated hydroxylation has recently been purported to function in such diverse roles as sympathetic neuronal and muscle development, sepsis, glycolytic metabolism, and cell fate. PHD3 expression is also highly distinct from that of the other PHD enzymes, and varies considerably between different cell types and oxygen concentrations. This thesis will specifically examine the role of PHD3 expression in cancer cells, with a focus on the mechanisms of PHD3 gene silencing. In the final chapters, I will examine the consequences of this silencing in cancer, and discuss the discovery of a novel role for PHD3 in epithelial-to-mesenchymal-like transition and cell migration.

<p>One of today’s most widely spread health problems is urinary incontinence, affecting 60-80% of the US population from age 15 and up. Treatment based on the possibility to implant a scaffold seeded with the patients’ own urine-derived stem cells, hUSC, to regenerate the damaged muscle tissue, would prove effective. A main challenge in regenerating new tissue from cell-seeded scaffolds is the limited cell survival due to insufficient oxygen diffusion to the center of the scaffold. Ways of enhancing cell survival, and thereby, proliferation and differentiation, is by hypoxic preconditioning of the cells or implantation in an oxygen-release material. Hypoxic preconditioning has shown to enhance proliferation as well as the expression of vascular endothelial growth factor, VEGF, in for example human bone marrow derived stem cells, hBMSC. VEGF is involved in the establishment of vasculature structures and an upregulation of its expression may therefore help promote quicker angeogenisis, increasing the oxygen supply and the cell survival. Oxygen-release materials have shown to enhance cell survival and growth both <em>in vitro</em> and <em>in vivo</em>.<em></em></p><p>This study aims to investigate the effect of hypoxia on hUSC, during 9 days of hypoxic culturing (2.0% ± 0.1% O₂) with and without oxygen-release material (PLGA 75:25 with 5 w% CPO) <em>in vitro</em>. hBMSC, and human smooth muscle cells, hSMC, have been used as control groups. Cell proliferation, morphology, differentiation, production of VEGF, and expression of hypoxia inducible factor HIF-1α have been studied.</p><p>According to the results, combining hypoxic preconditioning of hUSC with implantation in oxygen-release material could be an effective way to regenerate muscular tissue. Hypoxic preconditioning enhanced cell proliferation, production of VEGF, and HIF-1α expression. The increase of VEGF and HIF-1α would promote vascularization when implanted. The oxygen-release material showed possible promotion of cell differentiation, which would augment the hUSCs’ myogenic differentiation, while supplying oxygen until the tissue’s vascular structure has been established.</p>

Prostate cancer is the second most common cancer among men in the United States. While treatments for prostate cancer exist, none are curative. As a solid tumor, prostate cancer can grow beyond the diffusion limits of oxygen, thereby resulting in a hypoxic environment. While hypoxia can cause death to a variety of cell types, tumor cells can develop resistance to hypoxia and survive under minimal oxygen conditions. Hypoxia in tumor cells has also been associated with poor prognosis, increased metastasis, and decreased efficacy of chemotherapy. BNIP3, a BH-3 only proapoptotic Bcl-2 family member, has been shown to play an important role in cell death under hypoxic conditions in a variety of cell types. In normoxia, BNIP3 shows little to no expression in both cardiomyocytes and many cancer cell types, but is then upregulated under hypoxic conditions. Previous work in our laboratory provides evidence that hypoxia alone, as well as the concomitant increase in BNIP3 expression, cannot cause death of rat neonatal cardiomyocytes. Instead, our studies found that hypoxia with concomitant intracellular acidosis is required. Further studies indicated that BNIP3 is also necessary for hypoxia-acidosis associated cell death in cardiomyocytes. Our results in rat neonatal cardiomyocytes led us to hypothesize that cell death could be induced in hypoxic prostate cancer cells if concomitant acidosis could be induced. Additionally, our intention was to determine if BNIP3 was required for any prostate cancer cell death that may occur under hypoxia-acidosis conditions.