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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).

Die ständige Verfügbarkeit von molekularem Sauerstoff (O2) ist ein elementarer Bestandteil multizellulärer Lebensformen. Zur Aufrechterhaltung der Homöostase sind diese auf die Bildung des Energiesubstrates ATP durch oxidative Phosphorylierung angewiesen. Aus diesem Grunde mußten höhere Organismen während der Evolution komplexe Systeme entwickeln, die die Aufnahme und Verteilung von O2 in jede Zelle sicherstellen, sowie eine Adaptation in Phasen der Hypoxie erlauben. Mit der Identifikation des Transkriptionsfaktors "Hypoxia-inducible Factor-1" (HIF-1, 1995) wurde ein entscheidender Regulator der hypoxischen Adaptation gefunden. Unter anderem werden Prozesse wie die Erythropoiese, die Angiogenese, die Modulation des Gefäßtonus, des Glukosetransportes und der Glykolyse wesentlich durch HIF reguliert. HIF ist ein Heterodimer bestehend aus zwei Untereinheiten; einer konstitutiven beta- und einer regulativen alpha-Untereinheit. Letztere zeigt ein inverses Expressionsmuster zur perizellulären O2-Konzentration. Unter normoxischen Bedingungen ist HIFalpha instabil und wird mit einer Halbwertzeit von nur wenigen Minuten degradiert. Erst unter Hypoxie wird HIFalpha stabilisiert und ist transkriptionell aktiv. Es konnten bisher zwei funktionell relevante O2-abhängige alpha-Untereinheiten identifiziert werden: HIF-1 und HIF-2alpha. Die Bedeutung dieser beiden Systeme, der unterliegenden Regulationsmechanismen sowie die Relevanz dieses Systems in vivo waren weitgehend ungeklärt und sind wesentlicher Teil der hier zusammengefaßten Arbeiten. In den vorgelegten Studien ist es gelungen, die Expression und Regulation der beiden unterschiedlichen HIFalpha Isoformen sowohl in der Zellkultur, als auch in gesunden Geweben zu charakterisieren. In Zellkulturen zeigte sich ein sehr ähnliches Regulationsmuster hinsichtlich der O2-abhängigen Degradation, bzw. dem Induktionsverhalten unter Hypoxie, sowie der chemisch/pharmakologischen Modulation, so dass offensichtlich beide Isoformen über den gleichen O2-Sensing- und Transduktionsapparat reguliert werden. An Geweben von gesunden Ratten führten wir eine systematische Analyse der Expression und Regulation der beiden HIFalpha Isoformen durch. Nur unter systemischer Hypoxie konnten deutliche Signale für beide Isoformen gesehen werden. Interessanterweise zeigte sich, daß beide nur von spezifischen Zellpopulationen exprimiert werden. In vivo lassen sich also klare Unterschiede im Expressionsmuster der beiden Systeme feststellen. Über die unterschiedlichen zellulären Funktionen und different exprimierten Zielgene vermuten wir einen funktionell relevanten Unterschied. Mit der Identifikation des "von Hippel Lindau" Tumor Suppressor Gens als der bindende Anteil der E3 Ubiquitin Ligase, die für die HIFalpha Destruktion verantwortlich ist, konnte ein wichtiger Beitrag zu der späteren Klärung des O2-Sensing-Mechanismus geleistet werden. Diese Befunde wurden initial anhand von Zellkultur-Linien erhoben, liessen sich aber auf Nierenzellkarzinome aus einer klinischen Sammlung übertragen. Letzterer Befund ist daher für das Verständnis der Rolle von HIF für die Tumorbiologie, eventuell aber auch für die Entwicklung therapeutischer Ansätze von Bedeutung. / The permanent availability of molecular oxygen (O2) is an elemental need of multicellular life. For the maintenance of hemeostasis these are dependent on generation of the energy substrate ATP by oxidative phoshorylation. For this reason higher organisms had to develop complex systems during evolution that ensure the uptake and distribution of O2 into each cell, as well as permit adaptation to phases of hypoxia. With the identification of the transcription factor "Hypoxia-inducible Factor-1" (HIF-1, 1995) a master regulator of hypoxic adaptation has been found. Amongst others processes like erythropoiesis, angiogenesis, modulation of vascular tone, glucose transport and glycolysis are largely regulated by HIF. HIF is a heterodimer consisting of two subunits, a constitutive beta and a regulative alpha subunit. The latter shows an inverse relationship to the pericellular O2 concentration. HIFalpha is instable under normoxic conditions and degrades with a half life of only a few minutes. Under hypoxia the HIFalpha subunits are stabilised and are transcriptionally active. To date two functionally relevant O2-dependent alpha subunits have been identified: HIF-1 and HIF-2alpha. The importance of these two systems, the underlying regulatory mechanisms, as well as the relevance of this system in vivo were largely unknown and are a major part of the summarised studies. The presented work succeeded in characterising the expression and regulation of both HIFalpha isoforms in cell culture as well as healthy tissues. In tissue culture a very similar pattern of regulation was seen for oxygen dependent degradation, induction under hypoxia and chemical/pharmacological modulation, indicating that both subunits are regulated by the same O2-sensing and transduction apparatus. We undertook a systematic analysis of expression and regulation of both HIFalpha subunits in tissues of healthy rats. Signals for HIFalpha could only be seen under systemic hypoxia. Interestingly, both subunits were expressed by specific and different cell populations. Therefore, clear differences can be seen in expression pattern of both systems in vivo. We suspect that these differences will be functionally relevant through differing cellular functions and gene expression profile. With the identification of the "von Hippel Lindau" tumor suppressor gene as the binding part of the E3 ubiquitin ligase, which is responsible for HIF degradation, an important contribution to the clarification of the oxygen sensing mechanism was provided. Initially this data was generated in tissue culture lines, but could also be confirmed in renal cell carcinomas of a clinical collection. The latter finding is of importance for the understanding of the role of HIF in tumor biology, possibly also for the development of therapeutic strategies.

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