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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.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24801号 / 医博第4993号 / 新制||医||1067(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 鈴木 実, 教授 溝脇 尚志, 教授 江木 盛時 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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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.

Doctor of Philosophy / Department of Biochemistry / Dolores J. Takemoto / Connexin proteins are the principle structural components of gap junction channels that connect the cytoplasm of two cells and maintain direct intercellular communication through the exchange of ions, small molecules and cellular metabolites. Colocalization and tissue-specific expression of diverse connexin molecules are reported to occur in a variety of organs. Impairment of gap junctional intercellular communication, caused by mutations, gain of function or loss of function of connexins, is involved in a number of diseases including the development of cancer. Here the functions of a gap junction protein, connexin46 (Cx46), have been investigated in two hypoxic tissues, lens and breast tumor. We show that human breast cancer cells, MCF-7 and breast tumor tissues express connexin46 (Cx46) and it plays a critical role in protecting cells against hypoxia-induced death. Interestingly, I find that Cx46 is upregulated in MCF-7 breast cancer cells and human breast cancer tumors. Downregulation of Cx46 by siRNA promotes cell death of human lens epithelial cells (HLEC) and MCF-7 cells under hypoxic conditions. Furthermore, direct injection of anti-Cx46 siRNA into xenograft tumors prevents tumor growth in nude mice. Our result suggests that both normal hypoxic tissue (lens) and adaptive hypoxic tissue (breast tumor) utilize the same protein, Cx46, as a protective strategy against hypoxia. In the last part of the dissertation, we show that over expression of Cx46 induces the degradation of another connexin, connexin43, in rabbit lens epithelial NN1003A cells. Over expression of Cx46 increases ubiquitination of Cx43. Moreover, the Cx46-induced Cx43 degradation is counteracted by inhibitors of proteasome. Taken together, these data indicate that the degradation of Cx43, upon Cx46 over expression, is mediated by the ubiquitin-proteasome pathway. I also provide evidence that that C-terminal tail of Cx46 is essential to induce degradation of Cx43. Therefore, our study shows that Cx46 has a novel function in the regulation of Cx43 turnover in addition to its conventional role as a gap junction protein. This may contribute to protection from hypoxia in both the lens and tumors.

BACKGROUND:Diffuse axonal injury is a common consequence of traumatic brain injury (TBI) and often co-occurs with hypoxia, resulting in poor neurological outcome for which there is no current therapy. Here, we investigate the ability of the multifunctional compound erythropoietin (EPO) to provide neuroprotection when administered to rats after diffuse TBI alone or with post-traumatic hypoxia.METHODS:Sprague-Dawley rats were subjected to diffuse traumatic axonal injury (TAI) followed by 30minutes of hypoxic (Hx, 12% O2) or normoxic ventilation, and were administered recombinant human EPO-alpha (5000IU/kg) or saline at 1 and 24hours post-injury. The parameters examined included: 1) behavioural and cognitive deficit using the Rotarod, open field and novel object recognition tests / 2) axonal pathology (NF-200) / 3) callosal degradation (hematoxylin and eosin stain) / 3) dendritic loss (MAP2) / 4) expression and localisation of the EPO receptor (EpoR) / 5) activation/infiltration of microglia/macrophages (CD68) and production of IL-1beta.RESULTS:EPO significantly improved sensorimotor and cognitive recovery when administered to TAI rats with hypoxia (TAI+Hx). A single dose of EPO at 1hour reduced axonal damage in the white matter of TAI+Hx rats at 1day by 60% compared to vehicle. MAP2 was decreased in the lateral septal nucleus of TAI+Hx rats / however, EPO prevented this loss, and maintained MAP2 density over time. EPO administration elicited an early enhanced expression of EpoR 1day after TAI+Hx compared with a 7-day peak in vehicle controls. Furthermore, EPO reduced IL-1beta to sham levels 2hours after TAI+Hx, concomitant to a decrease in CD68 positive cells at 7 and 14days.CONCLUSIONS:When administered EPO, TAI+Hx rats had improved behavioural and cognitive performance, attenuated white matter damage, resolution of neuronal damage spanning from the axon to the dendrite, and suppressed neuroinflammation, alongside enhanced expression of EpoR. These data provide compelling evidence of EPO's neuroprotective capability. Few benefits were observed when EPO was administered to TAI rats without hypoxia, indicating that EPO's neuroprotective capacity is bolstered under hypoxic conditions, which may be an important consideration when EPO is employed for neuroprotection in the clinic.

The DNA damage response (DDR) is a complex signalling cascade triggered in response to stress, in an attempt to maintain genomic integrity. Components of this pathway, such as ATM-mediated signalling, have been proposed to act as a barrier in the early stages of tumourigenesis. Regions of low oxygen concentrations (hypoxia) occur in most solid tumours and are associated with a poor prognostic outcome. Here, we investigated the DDR induced following hypoxia-induced replication stress in an attempt to decipher the mechanism of ATM activation in response to physiological stresses that do not induce DNA damage. We hypothesized that hypoxia-mediated chromatin changes could impact on ATM signalling. We have characterised H3 methylation in response to hypoxia and found oxygen dependent changes in H3K9me3, including both global and replication fork associated increases in this histone modification. Importantly, we have found that decreases in H3K9me3 result in loss or attenuation of ATM activation. Notably, in a background of replication stress and increased H3K9me3, ATM inhibition or loss leads to accumulation of DNA damage and a significant decrease in replication rates in hypoxia. We propose that when replication stress occurs in the presence of hypoxia-induced chromatin changes, ATM activation is facilitated by the induction of H3K9me3. In this context, we propose a novel and stress specific role for ATM-mediated signalling in maintaining replication and preventing the generation of DNA breaks that may compromise genomic integrity. Moreover, the biological consequences of the hypoxia-induced chromatin context and in particular hypoxia-induced H3K9me3 include the repression of APAK, a negative regulator of p53. Activation of p53 is a key consequence of the hypoxia-induced DDR. Here we found that SETDB1, one of the H3 methyltransferases induced by hypoxia, mediates APAK repression. We propose that H3K9me3 plays a role in regulating APAK expression to allow optimal induction of p53 dependent apoptosis in hypoxic conditions suggesting a further role for H3K9me3 in facilitating DDR signalling in hypoxia. Together, these data suggest that the hypoxic chromatin context is critical for the role of the DDR as a barrier to tumourigenesis and predict that altering the chromatin landscape in combination with DNA damaging therapies would be efficacious in the treatment of hypoxic tumours.