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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La restauration rapide du flux sanguin est essentielle pour limiter l’étendue de l’infarctus myocardique mais elle est à l’origine de lésions irréversibles. Le préconditionnement ischémique à distance (RIPC) qui désigne l’application non invasive de brèves séquences d’ischémie/reperfusion au niveau d’un organe à distance du cœur peut prévenir la survenue de ces lésions de reperfusion. De nombreuses études suggèrent une implication de facteurs de transcription et de facteurs humoraux dans la cardioprotection induite par le RIPC, mais leurs identités restent inconnues. Dans la première partie du travail, nous avons donc étudié le rôle potentiel du facteur de transcription induit par l’hypoxie (HIF-1α) dans la phase précoce du RIPC. Nous avons ainsi démontré, en utilisant deux modèles expérimentaux : les souris transgéniques déficientes en HIF-1α et les rats traités par un inhibiteur pharmacologique de HIF-1α, que HIF-1α n’est pas indispensable pour cette phase du RIPC. Dans la deuxième partie de ce travail, nous avons essayé d’identifier, de façon directe cette fois, le ou les facteurs humoraux responsables de l’effet protecteur du RIPC. En se basant sur les résultats des études protéomiques démontrant une augmentation des concentrations plasmatiques d’apolipoprotéine A1 (Apo A1) suite au RIPC, nous avons alors cherché à mettre en évidence si cette protéine pourrait être un facteur circulant du RIPC. L’Apo A1, injectée directement avant la réalisation de l’infarctus chez le rat, était capable de reproduire l’effet cardioprotecteur et d’activer les mêmes voies de signalisation du RIPC. L’Apo A1 pourrait donc être un facteur humoral du RIPC. / Although early restoration of blood flow to the ischemic heart is essential to reduce the extent of myocardial infarction, reperfusion per se may cause irreversible tissue injury. Remote ischemic preconditioning (RIPC), the phenomenon whereby brief episodes of I/R are applied in distant tissues or organs, can protect the myocardium against reperfusion injuries. Several studies suggest that transcription factors and humoral mediators may be involved in RIPC mechanisms ; however the actual identity of these factors remains unknown. Therefore, in the first part of the study, we aimed to identify the role of hypoxia inducible factor (HIF-1α) in the acute phase of RIPC. We have thus demonstrated, using two animal models: partially HIF-1α-deficient mice and rats pretreated with a pharmacological inhibitor of HIF-1α, that HIF-1α is not crucial for this phase of RIPC. In the second part of the study, we have used a more direct approach and attempted to identify one or more humoral mediators of RIPC. Based on the results of proteomic studies showing an increase in plasmatic apolipoprotein A1 (Apo A1) levels following RIPC, we thus sought to determine if Apo A1 may constitute a blood-borne factor involved in RIPC’s protective effects. Our findings indicated that Apo A1 injected before myocardial infarction in rats acutely protected the heart, recapitulating RIPC-induced cardioprotection and that Apo A1 share some common signaling pathways with RIPC. Apo A1 may then be a humoral mediator of RIPC.

Dita Kašparová Chronická hypoxie a exprese genů 4 Abstract Adaptation to chronic hypoxia (CH) is characterized by a variety of functional changes in order to maintain metabolic and energy homeostasis. It has been known for many years that both humans and animals indigenous or adapted to high-altitude hypoxia are more tolerant to an acute ischemic injury of the heart. Cardioprotective mechanisms activated by adaptive responses to chronic hypoxia can be the result of altered transcriptional regulations in left ventricles. Here we report results from the gene expression profiling of adaptive responses in three models of chronically hypoxic heart. Adult male Wistar rats were exposed for 21 days to either continuous normobaric hypoxia (CCH; 10% O2) or CCH interrupted daily by 1-hour reoxygenation (RCH) or CCH interrupted daily by 16-hour (CIH). Cardiprotective effect of CCH adaptation is abolished by brief daily reoxygenation, RCH adaptation. In the present study, we aimed to determine myocardial mRNA expression of 19 candidate genes divided into three important groups: i) Hypoxia inducible factor (HIF1α) and its prolyl and asparaginyl hyroxylases (PHDs and FIH respectively, ii) Creatine kinase (CK) isoenzymes which play important role in energy homeostases of heart and iii) the group of main enzymatic...

Hematopoietic stem cells (HSCs) have historically been difficult to maintain ex vivo with many attempts to culture them in vitro by mimicking their natural biological environment. Providing a hypoxic environment is one way to achieve this goal and can be performed by using hypoxia stimulating compounds that inhibits the degradation of HIF1a which plays an important role in regulating hypoxia. For each sample 50 murine HSCs were isolated with fluorescence-activated cell sorting (FACS) and cultured with different concentrations of the hypoxia inducible compound Adaptaquin for 13 days followed by analysing with flow cytometry. The results showed an increase in proliferation of treated cells with the highest average total viable cell count for cells treated with 100 nM Adaptaquin of 4,70 ± 1,12 x 105 cells compared to the control which had 2,39 ± 0,76 x 105 cells. The HSC frequency was highest in the control samples with an average of 1,91 ± 0,42 % compared to the 5 mM treated samples with the highest average HSC frequency which was 1,52 ± 0,82 %. The biggest noticeable difference between the control and treated samples was seen when observing the total cell count. The difference in proliferation was on the other hand too small to see significant difference between the samples. The conclusion is that Adaptaquin did not have any significant impact on keeping the cells undifferentiated but could have a potential to be used as a compliment to other factors to maintain HSCs in vitro and to mimic its hypoxic biological environment. / Hematopoetiska stamceller (HSCs) har historiskt sett varit svåra att odla ex vivo och många försök har genomförts in vitro genom att efterlikna deras naturliga biologiska miljö. Att tillhandahålla en hypoxisk miljö är en metod för att uppnå detta och kan göras med användning hypoxi-stimulerande substanser som hämmar nedbrytningen av HIF1a som spelar en viktig roll i regleringen av hypoxi. För varje prov isolerades 50 murina HSCs med fluorescence-activated cell sorting (FACS) och odlades med olika koncentrationer av det hypoxi-inducerande ämnet Adaptaquin under 13 dagar följt av analys med flödescytometri. Resultaten visade en ökning i avseende på proliferationen hos behandlade celler där det högsta genomsnittliga totala antalet levande celler behandlade med 100 nM Adaptaquin som var 4,70 ± 1,12 x 105 celler jämfört med kontrollen som hade 2,39 ± 0,76 x 105 celler. HSC-frekvensen var högst i kontrollproverna med ett genomsnitt på 1,91 ± 0,42 % jämfört med proverna behandlade med 5 mM Adaptaquin som hade den högsta genomsnittliga HSC-frekvensen som låg på 1,52 ± 0,82 %. Den största synliga skillnaden mellan kontroll- och behandlingsprover var synlig när det observerade totala antalet celler jämfördes mellan behandlade prover som i genomsnitt hade fler totala celler. Skillnaden i proliferation var å andra sidan för liten för att se en signifikant skillnad mellan proverna. Slutsatsen är att Adaptaquin inte hade någon signifikant påverkan på att hålla HSCs odifferentierade men kan ha potential att användas som ett komplement till andra faktorer för att odla HSCs in vitro och efterlikna dess hypoxiska biologiska miljö.

BackgroundHead and neck squamous cell carcinoma (HNSCC) is a common cancer. Positron emission tomography (PET) with 18F-fluorodeoxyglucose (18F-FDG) is a widely used imaging modality in HNSCC.PurposeTo provide evident data about associations between 18F-FDG PET and histopathology in HNSCC.Material and MethodsThe MEDLINE database was screened for associations between maximum standard uptake values (SUVmax) derived from 18F-FDG PET and histopathological features in HNSCC up to May 2020. Only papers containing correlation coefficients between SUVmax and histopathology were acquired. Overall, 23 publications were collected.ResultsThe following correlations were calculated: KI 67: 12 studies (345 patients), pooled correlation coefficient (PCC): 0.23 (95% confidence interval [CI] 0.06–0.40); hypoxia-inducible factor-1α: eight studies (240 patients), PCC: 0.24 (95% CI 0.06–0.42); microvessel density: three studies (64 patients), PCC: 0.33 (95% CI 0.02–0.65); vascular endothelial growth factor: two studies (59 cases), PCC: 0.27 (95% CI 0.02–0.51); tumor suppressor protein p53: four studies (159 patients), PCC: 0.05 (95% CI –0.41 to 0.51); epidermal growth factor receptor: two studies (124 patients), PCC: 0.21 (95% CI 0.05–0.37); tumor cell count: three studies (67 patients), PCC: 0.18 (95% CI –0.06 to 0.42); tumor cell apoptosis: two studies (40 patients), PCC: 0.07 (95% CI = –0.85 to 0.99); B-cell lymphoma-2 protein: two studies (118 patients); PCC: 0.04 (95% CI –0.65 to 0.74); glucose-transporter 1: 10 studies (317 patients), PCC: 0.20 (95% CI 0.10–0.30).ConclusionSUVmax derived from 18F-FDG PET cannot reflect relevant histopathological features in HNSCC.

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Von Hippel-Lindau (VHL) syndrome is an autosomal dominant predisposition to cancer in neurological tissues, the kidneys, adrenal glands, pancreas, and liver, including neurological hemangioblastoma (HB), pheochromocytoma (PCC), pancreatic neuroendocrine tumors (PNET), pancreatic and renal cysts, and clear cell renal cell carcinoma (ccRCC). The disease process follows Knudson's two-hit model, requiring spontaneous loss or mutation of a normal VHL tumor suppressor allele to induce expression of the disease. VHL syndrome principally involves dysregulation of oxygen sensing pathways including the Hypoxia Inducible Factor (HIF)-Vascular Endothelial Growth Factor-A (VEGF-A) and HIF-Erythropoietin (EPO) pathways. RNA sequencing (RNA-Seq) data from our previously published experiments revealed a potentially novel VEGF-A splice variant with excision of the VEGF Receptor-1 (VEGFR-1)/Flt-1 binding domain, rendering this isoform resistant to native down-regulation. Additionally, phenotypic changes were observed in adult VHL mutant mice, specifically very red appearing extremities with prominently visible vasculature. In order to determine the etiology of this phenotype, we observed red blood cell count, Epo gene expression levels, and arterialization of the blood vessels in these experimental mice as compared to littermate controls. Current research into the VEGF-A isoform is ongoing in the lab, and preliminary evidence for the etiology of the apparent chronic erythema phenotype is inconclusive due to lack of experimental replicates due to COVID-19 quarantine orders. / Master of Science / Von Hippel-Lindau (VHL) syndrome is characterized by cancer development primarily in the brain and spinal cord, kidneys, adrenal glands, pancreas, and liver. VHL syndrome involves mutations which render the VHL gene dysfunctional. Since the VHL gene's normal role is one of preventing cancer development, sensing oxygen levels, and impacting blood vessel development, it follows that the loss of this gene results in tumor development with a rich blood vessel network. One of the downstream effectors of this process is a signaling molecule called Vascular Endothelial Growth Factor-A (VEGF-A). Our lab found a unique variant of VEGF-A, which may be overactive in the body in the setting of VHL disease. Additionally, we noted that our VHL mutant mice turned very red, and we sought to identify the biological cause of this phenomenon. In order to determine the cause of this redness, we studied red blood cell counts and their regulatory gene (Erythropoietin, EPO), as well as potential blood vessel abnormalities using high-power microscopy.

Thesis (Ph. D.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).

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