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[ES] Las células mesenquimales estromales (MSC) poseen una serie de cualidades inmunológicas, pro-angiogénicas y regenerativas que las convierten en un excelente candidato para el tratamiento de diversas patologías. A pesar de las pruebas contundentes obtenidas en modelos preclínicos que demuestran la actividad terapéutica de las MSC, los ensayos clínicos no han podido mostrar hasta ahora un beneficio consistente, probablemente debido a deficiencias metodológicas y a la falta de estandarización, así como a la variabilidad genética intrínseca a los estudios en humanos. Debido a ello, ha sido necesario profundizar en los mecanismos responsables del beneficio terapéutico y rediseñar las estrategias clínicas. En los últimos años, se ha observado que la reparación tisular mediada por las MSC se produce de forma paracrina, y se ha constatado que las vesículas extracelulares (EVs) secretadas por las MSC (EVMSC) son capaces de recapitular las propiedades inmunosupresoras de las células parentales. Además, las estrategias terapéuticas basadas en vesículas tienen grandes ventajas en términos de bioseguridad y producción en condiciones de grado clínico, reduciendo significativamente el coste de dichas terapias. Sin embargo, la dosis efectiva en grandes mamíferos, incluidos los humanos, es bastante elevada y la producción industrial de EVs se ve dificultada en parte, por la senescencia proliferativa que afecta a las MSC durante la expansión celular masiva. En este trabajo hemos intentado solventar los principales escollos de la terapia con EVs incrementando su potencial inmunosupresor y reduciendo por tanto la dosis efectiva. Este incremento se ha conseguido gracias a la sobreexpresión del factor inducible por hipoxia 1-alpha y al desarrollo de un medio de acondicionamiento en cultivo basado en citoquinas. Además, la inmortalización de las células secretoras mediante la transducción del gen de la telomerasa humana ha permitido tanto la estandarización del producto como su producción a gran escala. La eficacia de estas EVs ha sido testada en diferentes poblaciones celulares in vitro: linfocitos T, monocitos, células Natural Killer, macrófagos, células endoteliales y fibroblastos; y en dos modelos de ratón: hipersensibilidad retardada y colitis aguda inducida por TNBS. En conclusión, hemos desarrollado una fuente de EVs de larga duración que secreta grandes cantidades de vesículas con mayor capacidad inmunosupresora y antiinflamatoria, facilitando un producto terapéutico más estándar y fácil de producir para el tratamiento de enfermedades inflamatorias inmunomediadas. / [CA] Les cèl·lules mesenquimals estromals (MSC) posseeixen una sèrie de qualitats immunològiques, pro-angiogèniques i regeneratives que les converteixen en un excel·lent candidat per al tractament de diverses patologies. Tot i les proves contundents obtingudes en models preclínics que demostren l'activitat terapèutica de les MSC, els assaigs clínics no han pogut mostrar fins ara un benefici consistent, probablement degut a deficiències metodològiques i a la manca d'estandardització, així com a la variabilitat genètica intrínseca als estudis en humans. A causa d'això, ha calgut aprofundir en els mecanismes responsables del benefici terapèutic i redissenyar les estratègies clíniques. En els últims anys, s'ha observat que la reparació tissular intervinguda per les MSC es produeix de forma paracrina, i s'ha constatat que les vesícules extracel·lulars (EVs) secretades per les MSC (EVMSC) són capaços de recapitular les propietats immunosupressores de les cèl·lules parentals. A més, les estratègies terapèutiques basades en vesícules tenen grans avantatges en termes de bioseguretat i producció en condicions de grau clínic, reduint significativament el cost d'aquestes teràpies. No obstant això, la dosi efectiva en grans mamífers, inclosos els humans, és bastant elevada i la producció industrial de les EVs es veu dificultada en part, per la senescència proliferativa que afecta les MSC durant l'expansió cel·lular massiva. En aquest treball hem intentat solucionar els principals esculls de la teràpia amb EVs incrementant el seu potencial immunosupressor i reduint per tant la dosi efectiva. Aquest increment s'ha aconseguit gràcies a la sobreexpressió del factor induïble per hipòxia 1-alpha i a el desenvolupament d'un mitjà de condicionament en cultiu basat en citoquines. A més, la immortalització de les cèl·lules secretores mitjançant la transducció del gen de la telomerasa humana ha permès tant l'estandardització del producte com la seva producció a gran escala. L'eficàcia d'aquestes EVs ha estat testada en diferents poblacions cel·lulars in vitro: limfòcits T, monòcits, cèl·lules Natural Killer, macròfags, cèl·lules endotelials i fibroblasts; i en dos models de ratolí: hipersensibilitat retardada i colitis aguda induïda per TNBS. En conclusió, hem desenvolupat una font de EVs de llarga durada que secreta grans quantitats de vesícules amb major capacitat immunosupressora i antiinflamatòria, facilitant un producte terapèutic més estàndard i fàcil de produir per al tractament de malalties inflamatòries inmunomediades. / [EN] Mesenchymal stromal cells (MSC) possess several immunological, pro-angiogenic and regenerative qualities that make them an excellent candidate for the treatment of various pathologies. Despite compelling evidence from preclinical models demonstrating the therapeutic activity of MSCs, clinical trials have so far failed to show consistent benefit, probably due to methodological shortcomings and lack of standardisation, as well as the genetic variability intrinsic to human studies. As a result, it has been necessary to further investigate the mechanisms responsible for therapeutic benefit and to redesign clinical strategies. In recent years, it has been observed that MSC-mediated tissue repair occurs in a paracrine pathway, and it has been confirmed that extracellular vesicles (EVs) secreted by MSC (EVMSC) are able to recapitulate the immunosuppressive properties of the parental cells. Moreover, vesicle-based therapeutic strategies have great advantages in terms of biosafety and production under clinical-grade conditions, significantly reducing the cost of such therapies. However, the effective dose in large mammals, including humans, is quite high and the industrial production of EVs is hampered in part by the proliferative senescence that affects MSC during massive cell expansion. In this work, we have attempted to overcome the main challenges of EVs therapy by increasing their immunosuppressive potential and thus reducing the effective dose. This increase has been achieved by overexpression of hypoxia-inducible factor 1-alpha and the development of a cytokine-based culture conditioning medium. In addition, immortalization of secretory cells by transduction with the human telomerase gene has allowed both product standardisation and large-scale production. The efficacy of these EVs has been tested in different cell populations in vitro: T lymphocytes, monocytes, Natural Killer cells, macrophages, endothelial cells and fibroblasts; and in two mouse models: delayed-type hypersensitivity and TNBS-induced acute colitis. In conclusion, we have developed a long-lasting source of EVs that secretes large amounts of vesicles with enhanced immunosuppressive and anti-inflammatory capacity, providing a more standard and easier-to-produce therapeutic product for the treatment of immune-mediated inflammatory diseases. / Gómez Ferrer, M. (2022). Terapias innovadoras basadas en vesículas extracelulares derivadas de células madre mesenquimales modificadas genéticamente [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/182560

Lípides nitrados (NO2-FA) são apontados como uma nova classe de mediadores lipídicos, podendo atuar como reservatórios endógenos de óxido nítrico (&#8226NO) bem como moduladores pluripotentes de sinalização celular. Recentemente, tem sido sugerido que os doadores de &#8226NO estariam envolvidos na regulação da angiogênese. Evidências contundentes indicam ainda que o processo de neovascularização poderia contribuir para a patogênese de uma serie de condições clínicas, entre elas a aterosclerose. Contudo, apesar de diversos estudos terem explorado os efeitos biológicos dos NO2-FA, os efeitos destes compostos sobre o processo de angiogênese não haviam sido descritos. Dessa maneira, o presente trabalho investigou os efeitos dos NO2-FA (derivados da nitração do ácido linoléico e oléico) noprocesso de angiogênese. Demonstrou-se que os NO2-FA podem atuar como mediadores pró-angiogênicos. Este efeito foi caracterizado em células endoteliais humanas, assim como, em modelos ex vivo e in vivo. Nas células endoteliais, observou-se que os No2-FA não influenciaram a proliferação ou a viabilidade celular, ao passo que estimularam a migração. Demonstrou-se também que os NO2-FA podem modular o brotamento ex vivo de novos vasos, em cultura de anéis de aorta de rato, bem como o processo angiogênico in vivo observado na membrana corioalantóica de embrião de galinha. Adicionalmente, os NO2-FA induziram a expressão do fator de crescimento endotelial vascular (VEGF), que é o principal mediador do processo de angiogênese. Em relação ao mecanismo de ação, os achados sugerem que os efeitos demonstrados seriam via mecanismos dependentes de &#8226NO, uma vez que foram abolidos na presença de um seqüestrador de &#8226NO, enquanto concentrações equivalentes dos lípides precursores não demonstraram qualquer influência nas condições experimentais utilizadas neste estudo. Por fim, os efeitos pró-angiogênicos dos NO2-FA foram mediados pela estabilização da proteína do fator induzível por hipóxia -1α (HIF-1α), uma vez que estes compostos promoveram acúmulo desta proteína e falharam em demonstrar efeitos indutores em células knockdown para o gene HIF-1α. Em conjunto, estes resultados indicam que os NO2-FA podem modular a migração de células endoteliais e estimular o processo de angiogênese resultante da ativação de HIF-1a via mecanismo dependente de &#8226NO. / Nitrated lipids (NO2-FA) are described as a new class of Iipid mediators that are able to act as endogenously nitric oxide (&#8226NO) reservoirs as well as pluripotent cell signaling modulators. Furthermore, recent findings suggest that &#8226NO donors could be involved in the regulation of angiogenesis. Compelling evidence also indicate that the neovascularization process might contribute to the pathogenesis of many clinical conditions, such as atherosclerosis. However, although several studies have explored the NO2-FA biological properties, the effects of these compounds on the angiogenic process remain unknown. Hence, the present study investigated the effects of the NO2-FA (derivates from the nitration of Iinoleic and oleic acids at physiological concentrations) on angiogenesis processo It is demonstrated that the No2-FA could act as pro-angiogenic mediators. This effect was observed not only in human endothelial cells but also in ex vivo and in vivo models. Using endothelial cells, it is showed that NO2-FA failed to affect cell proliferation ar influence cellular viability, but significantly stimulated cell migration. It was also found that the NO2-FA might modulate the ex vivo sprouting of new vessels as well as the in vivo angiogenic process, while inducing the expression of the vascular endothelial growth factor, the main mediator of angiogenesis. The data are consistent with the hypothesis that the observed effects mediated by NO-dependent mechanisms, since the presence of a &#8226NO scavenger abrogated the induced effects, whereas equimolar concentrations of its precursors, showed no effect on angiogenesis under our experimental conditions. Finally, the pro-angiogenic effects of NOrFA were mediated by the stabilization of the hypoxia inducible factor-1α (HIF-1α) protein, because these compounds increased the protein amount and failed to show inductive effects in HIF-1α knockdown cells. Taken together, these findings indicated that NO2-FA might modulate the endothelial cell migration and stimulate the process of angiogenesis by the HIF-1α induction through a &#8226NO-dependent mechanism.

Lípides nitrados (NO2-FA) são apontados como uma nova classe de mediadores lipídicos, podendo atuar como reservatórios endógenos de óxido nítrico (&#8226NO) bem como moduladores pluripotentes de sinalização celular. Recentemente, tem sido sugerido que os doadores de &#8226NO estariam envolvidos na regulação da angiogênese. Evidências contundentes indicam ainda que o processo de neovascularização poderia contribuir para a patogênese de uma serie de condições clínicas, entre elas a aterosclerose. Contudo, apesar de diversos estudos terem explorado os efeitos biológicos dos NO2-FA, os efeitos destes compostos sobre o processo de angiogênese não haviam sido descritos. Dessa maneira, o presente trabalho investigou os efeitos dos NO2-FA (derivados da nitração do ácido linoléico e oléico) noprocesso de angiogênese. Demonstrou-se que os NO2-FA podem atuar como mediadores pró-angiogênicos. Este efeito foi caracterizado em células endoteliais humanas, assim como, em modelos ex vivo e in vivo. Nas células endoteliais, observou-se que os No2-FA não influenciaram a proliferação ou a viabilidade celular, ao passo que estimularam a migração. Demonstrou-se também que os NO2-FA podem modular o brotamento ex vivo de novos vasos, em cultura de anéis de aorta de rato, bem como o processo angiogênico in vivo observado na membrana corioalantóica de embrião de galinha. Adicionalmente, os NO2-FA induziram a expressão do fator de crescimento endotelial vascular (VEGF), que é o principal mediador do processo de angiogênese. Em relação ao mecanismo de ação, os achados sugerem que os efeitos demonstrados seriam via mecanismos dependentes de &#8226NO, uma vez que foram abolidos na presença de um seqüestrador de &#8226NO, enquanto concentrações equivalentes dos lípides precursores não demonstraram qualquer influência nas condições experimentais utilizadas neste estudo. Por fim, os efeitos pró-angiogênicos dos NO2-FA foram mediados pela estabilização da proteína do fator induzível por hipóxia -1α (HIF-1α), uma vez que estes compostos promoveram acúmulo desta proteína e falharam em demonstrar efeitos indutores em células knockdown para o gene HIF-1α. Em conjunto, estes resultados indicam que os NO2-FA podem modular a migração de células endoteliais e estimular o processo de angiogênese resultante da ativação de HIF-1a via mecanismo dependente de &#8226NO. / Nitrated lipids (NO2-FA) are described as a new class of Iipid mediators that are able to act as endogenously nitric oxide (&#8226NO) reservoirs as well as pluripotent cell signaling modulators. Furthermore, recent findings suggest that &#8226NO donors could be involved in the regulation of angiogenesis. Compelling evidence also indicate that the neovascularization process might contribute to the pathogenesis of many clinical conditions, such as atherosclerosis. However, although several studies have explored the NO2-FA biological properties, the effects of these compounds on the angiogenic process remain unknown. Hence, the present study investigated the effects of the NO2-FA (derivates from the nitration of Iinoleic and oleic acids at physiological concentrations) on angiogenesis processo It is demonstrated that the No2-FA could act as pro-angiogenic mediators. This effect was observed not only in human endothelial cells but also in ex vivo and in vivo models. Using endothelial cells, it is showed that NO2-FA failed to affect cell proliferation ar influence cellular viability, but significantly stimulated cell migration. It was also found that the NO2-FA might modulate the ex vivo sprouting of new vessels as well as the in vivo angiogenic process, while inducing the expression of the vascular endothelial growth factor, the main mediator of angiogenesis. The data are consistent with the hypothesis that the observed effects mediated by NO-dependent mechanisms, since the presence of a &#8226NO scavenger abrogated the induced effects, whereas equimolar concentrations of its precursors, showed no effect on angiogenesis under our experimental conditions. Finally, the pro-angiogenic effects of NOrFA were mediated by the stabilization of the hypoxia inducible factor-1α (HIF-1α) protein, because these compounds increased the protein amount and failed to show inductive effects in HIF-1α knockdown cells. Taken together, these findings indicated that NO2-FA might modulate the endothelial cell migration and stimulate the process of angiogenesis by the HIF-1α induction through a &#8226NO-dependent mechanism.

Abstract Mediators of epithelial permeability, angiogenesis and invasion may serve as prognostic indicators and targets for therapies in esophageal cancer (EC). The expressions of claudins, hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor-A (VEGF), nitric oxide synthases (iNOS, eNOS, nNOS) and toll-like receptor 9 (TLR9) were evaluated by immunohistochemistry in EC. The results were compared with clinicopathological variables, tumor proliferation and apoptosis. All of the claudins were expressed in most of the cancers. Esophageal adenocarcinomas (EAC) displayed more often increased claudin 3 and 5 expression than esophageal squamous cell carcinomas (ESCC). Loss of claudin 3 expression associated with distant metastases in EC and a tendency in this direction was also observed for claudin 4. Cancers with stronger claudin 4 expression showed increased apoptosis in both EAC and ESCC. HIF-1α was present in most of the ECs and like iNOS more often in ESCC than in EAC. Strong HIF-1α expression tended to associate with positive VEGF immunostaining. In ESCC, both strong HIF-1α expression and VEGF positivity tended to associate with increased microvessel density (MVD). In EAC, tumors showing VEGF positivity associated with increased MVD outside the tumor. Patients with strong HIF-1α expression had more often distant metastases than other patients in EC. There was no VEGF expression in normal esophageal mucosa and T3-4 tumors tended to be more often VEGF positive than T1-2 tumors. The expression of TLR9 was more intensive in dysplasia than in normal epithelium and ESCC and abundant TLR9 expression could serve as a marker of squamous cell high grade dysplasia. Intensive TLR9 expression was associated with higher grade tumors and the presence of nodal and distant metastases in ESCC. EC and its progression may be related to increased angiogenesis regulated by VEGF and HIF-1α. In EC, claudin expression varies along with the histology of the tumor. Claudin expression may be associated with apoptosis or proliferation and contribute to tumor behavior. An association was detected between moderate to strong expression of claudin 3 and a high TLR9 histoscore in ESCC. Altered expression of claudin 3 may result to upregulation of endosomal TLR9. TLR9 may serve as a marker for squamous cell dysplasia and ESCC progression. / Tiivistelmä Solukerrosten läpäisevyyttä, verisuonten uudismuodostusta ja kasvainsolujen liikkuvuutta säätelevät tekijät voivat toimia ruokatorvisyövän ennustetekijöinä ja hoidon kohteina. Tässä tutkimuksessa selvitettiin klaudiinien, hypoksia-indusoituvan tekijän 1α (HIF-1α), verisuonen endoteelin kasvutekijän A (VEGF), kolmen typpioksidisyntaasin (iNOS, eNOS ja nNOS) sekä tollin kaltaisen reseptorin 9 (TLR9) ilmentymistä ja merkitystä ruokatorvisyövässä immunohistokemiallisin menetelmin. Tuloksia arvioitiin suhteessa proliferaatioon ja apoptoosiin. Useimmat syöpäkasvaimet ilmensivät jokaista tutkittua klaudiinia. Ruokatorven rauhassyövässä ilmeni levyepiteelisyöpää useammin klaudiineja 3 ja 5. Klaudiinin 3 vähäinen ilmentyminen oli yhteydessä etäpesäkkeiseen tautiin. Sama suuntaus näkyi klaudiinin 4 kohdalla. Apoptoosia todettiin enemmän kasvaimissa, jotka ilmensivät muita enemmän klaudiini 4:ä. Useimmat syöpäkasvaimet ilmensivät HIF-1α:a. Sekä iNOS että HIF-1α ilmentyivät runsaammin levyepiteeli- kuin rauhassyövässä. Tutkimus viittasi yhteyteen voimakkaan HIF-1α:n ilmentymisen ja VEGF:n ilmentymisen välillä. Voimakas HIF-1α:n ilmentyminen ja VEGF:n ilmentyminen vaikuttivat liittyvän ruokatorven levyepiteelisyövän uudissuonituksen lisääntymiseen. Rauhassyövän lisääntynyt uudissuonitus kasvaimen ulkopuolella saattaa liittyä VEGF:n ilmentymiseen. Potilailla, joiden kasvaimissa HIF-1α:n ilmentyminen oli voimakasta, todettiin etäpesäkkeitä muita useammin. VEGF:a ei todettu normaalissa limakalvossa, ja sen ilmentyminen vaikutti olevan yleisempää syvälle kasvavissa kuin pinnallisissa syövissä. TLR9 ilmentyi voimakkaammin levyepiteelin dysplasiassa kuin normaalissa tai kasvainepiteelissä. Huonosti erilaistuvissa ja levinneissä kasvaimissa TLR9 ilmentyi voimakkaammin kuin muissa kasvaimissa. Ruokatorvisyövän synty ja eteneminen voivat liittyä HIF:n ja VEGF:n säätelemään verisuonten uudismuodostukseen. Klaudiinit saattavat vaikuttaa syövän käyttäytymiseen myös apoptoosin ja proliferaation kautta. Tutkimuksessa todettiin yhteys lisääntyneen klaudiinin 3 ilmentymisen ja voimakkaasti ilmentyvän TLR9:n välillä. Muutos klaudiinin 3 ilmentymisessä saattaa lisätä levyepiteelin läpäisevyyttä johtaen TLR9:n aktivoitumiseen. TLR9 voi vaikuttaa ruokatorven levyepiteelin dysplasian ja syövän syntyyn sekä toimia vaikean dysplasian ja aggressiivisen levyepiteelisyövän merkkiaineena.

The aryl hydrocarbon receptor (AhR) heterodimerizes with the aryl hydrocarbon receptor nuclear translocator (Arnt) for transcriptional regulation. We generated three N-terminal deletion constructs of the human AhR of 12-24 KDa in size—namely D1 (aa 84-295), D2 (aa 84-192) and D3 (aa 191-295)—to suppress the Arnt function. We observed that all three constructs interact with the human Arnt with similar affinities. D2, which contains part of the AhR PAS-A domain and interacts with the PAS-A domain of Arnt, inhibits the formation of the AhR gel shift complex. D2 suppresses the 3-methylcholanthrene-induced, dioxin response element (DRE)-driven luciferase activity in Hep3B cells and exogenous Arnt reverses this D2 suppression. D2 suppresses the induction of CYP1A1 at both the message and protein levels in Hep3B cells; however, the CYP1B1 induction is not affected. D2 suppresses the recruitment of Arnt to the cyp1a1 promoter but not to the cyp1b1 promoter, partly because the AhR/Arnt heterodimer binds better to the cyp1b1 DRE than to the cyp1a1 DRE. Interestingly, D2 has no effect on the cobalt chloride-induced, hypoxia inducible factor-1 (HIF-1)-dependent expression of vegf, aldolase c, and ldh-a messages. Our data reveal that the flanking sequences of the DRE contribute to the binding affinity of the AhR/Arnt heterodimer to its endogenous enhancers and the function of AhR and HIF-1 can be differentially suppressed by the D2 inhibitory molecule. In chapter 2, a Pichia Pastoris expression system was constructed expressing codon optimized human full length AhR. This codon optimization is necessary for overexpression of huAhR. RT-PCR data showed that the codon optimized mRNA was more stably expressed than wild types. Overexpressed huAhR protein was degraded by proteinase when using a regular P. Pastoris strain yJC100 whereas the proteinase deficient ySMD1163 maintained a much higher level of huAhR. P. Pastoris expressed huAhR was natively purified and analyzed. Coimmunopricipitation assay shows its interaction with endogenous Arnt. A ligand-dependent gel shift was also observed. In addition, we performed an in vitro coprecipitation assay to study its binding to endogenous cyp1b1 DREs. The result shows that the DRE3, known as a critical DRE for cyp1b1 transcriptional activity, has the highest binding affinity to AhR/Arnt complex. Taking together, we constructed a novel P. Pastoris expression system to overexpress human full length AhR. Purified huAhR is a good reagent for studing its ligand and DNA binding. In chapter 3, an adeno-associated virus (AAV) expression system was constructed to express an AhR deletion contruct CΔ553 (aa1-295) for tumor injection. Western blot shows the expression of CΔ553 (aa1-295) in hela cells infected by AAV-553, but the low yield of AAV-553 limited its application on tumor treatment. Possible solutions were discussed for future work.

<p> Exposure to chronic low oxygen (hypoxia) leads to a series of adaptive responses involving changes in gene expression that are critical for cell, tissue, and organismal survival. These changes are mediated by an important set of regulators belonging to the hypoxia inducible factor (HIF) family of transcription factors (e.g. HIF-lα, HIF-2α, HIF3α) which undergo rapid degradation during normal oxygen (normoxia) but are rapidly stabilized during hypoxia. While the role of HIF-1α has been extensively studied in many cell types, there have been relatively few studies on the role of HIF-2α, though recent evidence suggests its function maybe tissue specific. This thesis examined the hypothesis that HIF-2α plays a central role in the development and function of catecholaminergic cells of the sympathoadrenal (SA) lineage. The study was aided by use of an immortalized line of rat adrenomedullary chromaffin cells (i.e. MAH cells), derived from fetal SA progenitors, which express several hypoxia-sensitive properties characteristic of native cells in the adrenal gland. In Chapter 2, I investigated the potential contributions of mitochondrial reactive oxygen species (ROS) and 0 2 consumption to HIF-2α induction in MAH cells exposed to chronic hypoxia (2% O(2); 24 hr). In MAH cells, chronic hypoxia caused an increase in HIF-2α induction which was blocked by inhibition of any of the mitochondrial complexes using pharmacological agents, or by specific inhibition of complexes III and IV using RNAi techniques. It was found that in this 0 2-sensitive chromaffin cell line mitochondrial O(2) consumption, rather than changes in ROS, regulated HIF-2α induction during hypoxia. In Chapter 3, I investigated the hypothesized role of HIF-2α in the development of the catecholaminergic phenotype in cells of the SA lineage using the MAH cell line as a model. Mutant MAH cells, with depleted HIF-2α due to siRNA knock-down, showed dramatically lower levels of dopamine and noradrenaline compared to untransfected and scrambled control cells, regardless of whether the cells were cultured under normoxia or chronic hypoxia. This was correlated with a marked reduction in the expression of DOPA decarboxylase (DDC) and dopamine B hydroxylase (DBH), though the expression of tyrosine hydroxylase (TH) was unaffected. Moreover, HIF-2α was able to bind to a region of the DDC gene promoter which contains two putative hypoxia response elements (HREs). These data suggest that a basal level of HIF-2α function is required for the normal developmental expression of DDC and DBH in SA progenitor cells, and that loss of this function leads to impaired catecholamine (CA) biosynthesis. In Chapter 4, I investigated genes regulated by chronic hypoxia in MAH cells, with a focus on those involved in CA metabolism, storage, and secretion. Using microarray analysis combined with QPCR and RNAi knock-down methodology I uncovered several genes, involved in amine vesicular packaging, trafficking and secretion, which were upregulated during chronic hypoxia. One gene specifically, the adenosine A(2A) receptor (A(2A)R) gene, which appears to modulate CA secretion via autocrine or paracrine actions of extracellular adenosine, was dramatically upregulated in chronic hypoxia. Interestingly, this effect was completely abolished in HIF-2α knockdown MAH cells, suggesting a critical involvement of HIF-2α. Chromatin immunoprecipitation (ChIP) assays revealed that HIF-2α bound to the promoter region of the A(2A)R gene which contains a putative hypoxia response element (HRE) immediately upstream of exon 1. Ratiometric fluorescence measurements of intracellular Ca(2+) revealed that adenosine (50 μM) potentiated the high K(+)-evoked rise in [Ca(2+)]i in MAH cells. This effect of adenosine was further enhanced after chronic hypoxia, but was abolished in HIF-2α knock-down cells. In conclusion, these data suggest that HIF-2α is a key regulator of several genes involved in CA biosynthesis, and of others that mediate the facilitatory effects of chronic hypoxia on CA secretion in sympathoadrenal derivatives. / Thesis / Doctor of Philosophy (PhD)

Introdução: Existem dados suficientes disponíveis demonstrando a importância da quimiocina CXCL12 e seu receptor CXCR4 na progressão tumoral e angiogênese dos gliomas. O CXCR4 é regulado positivamente pelo HIF1alfa. Recentemente um novo receptor com maior afinidade à CXCL12 foi identificado, o receptor órfão RDC1, agora denominado CXCR7. O objetivo deste estudo é investigar a expressão de mRNA CXCR7 em tecidos astrocitomas difusos e avaliar suas interações com expressão CXCR4 e HIF1alfa, bem como analisar sua relação com mutação do IDH1. Métodos: A expressão do CXCR7, CXCR4, IDH1 e HIF1alfa foram avaliadas por PCR quantitativo em tempo real (qRT-PCR) em 129 amostras congeladas de astrocitomas (25 astrocitoma difuso - AGII, 18 de astrocitoma anaplásico - AGIII e 86 glioblastoma - GBM) e 22 amostras de tecido cerebral não neoplásico (NN) obtidos de cirurgia de epilepsia. A mutação do IDH1 previamente determinada foi analisada em relação aos níveis de expressões de mRNA do CXCR7, CXCR4 e HIF1alfa, combinado com os parâmetros clínico-patológicos e sobrevida global. Adicionalmente, a expressão proteica do CXCR7 foi analisada por imuno-histoquímica em astrocitomas de diferentes graus e em linhagem celular de glioma (U87MG) por microscopia confocal. Resultados: Houve diferença significativa nos níveis de expressão dos genes estudados entre astrocitomas e NN (p < 0,001). Na análise da expressão gênica associada nos AGII não se observou correlação entre os níveis de expressão de CXCR7/HIF1alfa (p = 0,548); observou-se correlação significativa entre CXCR7/IDH1 (p < 0,001) e CXCR7/CXCR4 (p = 0,042). Nos GBM houve correlação significativa entre CXCR7/CXCR4 (p = 0,002), CXCR7/IDH1 (p < 0,001) e CXCR7/HIF1alfa (p = 0,008). Hiperexpressão do HIF1alfa foi associado com maior expressão do CXCR7 e CXCR4 (p = 0,001), enquanto a presença de IDH1 mutado foi associada a menor expressão de mRNA do CXCR7 e CXCR4 (p = 0,009). A expressão proteica de CXCR7 foi identificada em todas as amostras estudadas, e aumentou com malignidade. A proteína CXCR7, na linha celular U87MG, foi localizada principalmente na membrana celular. Conclusão: O CXCR7 é um gene diferencialmente expresso em astrocitomas difusamente infiltrativos em relação tecido cerebral não neoplásico. O nível de expressão do CXCR7 correlacionou-se significativamente com os níveis de expressão do CXCR4 e IDH1 nos AGII e com CXCR4, IDH1 e HIF-1alfa nos GBM. O nível de expressão elevado do CXCR7 e CXCR4 correlacionou-se com nível elevado de expressão de HIF-1a, enquanto a presença da mutação do IDH1 associou-se a níveis reduzidos de CXCR7 e CXCR4. Não se observou associação significativa entre os níveis de expressão de CXCR7 e CXCR4 com os dados de sobrevida / Introduction: There is abundant evidence showing that chemokine CXCL12 and its receptor CXCR4 are involved in glioma progression and angiogenesis. CXCR4 is upregulated by HIF1alfa. The CXCR7, a recent additional receptor for CXCL12 with higher affinity than CXCR4 has raised key issues on glioma cell migration. The aim of this study is to investigate the CXCR7 mRNA expression in diffuse astrocytoma tissues and to evaluate its interactions with CXCR4 and HIF1alfa expression and IDH1 mutation. Methods: CXCR7, CXCR4, IDH1 and HIF1alfa expressions were evaluated by quantitative real-time PCR (qRT-PCR) in 129 frozen samples of astrocytoma (25 diffuse astrocytomas - AGII, 18 anaplastic astrocytomas - AGIII and 86 glioblastomas - GBM) and 22 samples of non-neoplastic tissue cerebral (NN) from epilepsy surgery. IDH1 mutation status was analyzed with CXCR7, CXCR4 e HIF1alfa mRNA expressions, matched with clinicopathological parameters and overall survival time. Furthermore, CXCR7 protein expression was analyzed by immunohistochemistry in different grades of astrocytoma and in glioma cell line (U87MG) by confocal microscopy. Results: There was significant difference in the expression levels of the genes studied between astrocytomas and NN (p < 0.001). The analysis of associated gene expressions in AGII showed no significant correlation between CXCR7/HIF1alfa (p = 0.548); there was significant correlation between CXCR7/CXCR4 (p = 0.042) and CXCR7/IDH1 (p = 0.008). In GBM, there were significant correlations between CXCR7/CXCR4 (p = 0.002), CXCR7/IDH1 (p < 0.001) and CXCR7/HIF1alfa (p = 0.008). HIF1alfa overexpression was associated with higher expressions of CXCR7 and CXCR4 (p = 0.001), while presence of IDH1 mutation was associated with lower CXCR7 and CXCR4 mRNA expressions (p = 0.009). Protein expression was identified in all samples studied, and it increased with malignancy. CXCR7 protein, in U87MG cell line, was mainly localized in the cellular membrane. Conclusion: CXCR7 was overexpressed in astrocytoma of different grades of malignancy compared to non-neoplastic brain tissue. CXCR7 expression levels correlates with CXCR4 and IDH1 in AGII and CXCR4, IDH1 and HIF1alfa in GBM. Overexpression HIF1alfa was related with higher expressions of CXCR7 and CXCR4, otherwise presence of IDH1 mutation related with lower expression of both genes. Protein expression level was associated with the degree of malignancy. The results revealed no significant association between CXCR7 and CXCR4 expression and survival data

Introdução: Existem dados suficientes disponíveis demonstrando a importância da quimiocina CXCL12 e seu receptor CXCR4 na progressão tumoral e angiogênese dos gliomas. O CXCR4 é regulado positivamente pelo HIF1alfa. Recentemente um novo receptor com maior afinidade à CXCL12 foi identificado, o receptor órfão RDC1, agora denominado CXCR7. O objetivo deste estudo é investigar a expressão de mRNA CXCR7 em tecidos astrocitomas difusos e avaliar suas interações com expressão CXCR4 e HIF1alfa, bem como analisar sua relação com mutação do IDH1. Métodos: A expressão do CXCR7, CXCR4, IDH1 e HIF1alfa foram avaliadas por PCR quantitativo em tempo real (qRT-PCR) em 129 amostras congeladas de astrocitomas (25 astrocitoma difuso - AGII, 18 de astrocitoma anaplásico - AGIII e 86 glioblastoma - GBM) e 22 amostras de tecido cerebral não neoplásico (NN) obtidos de cirurgia de epilepsia. A mutação do IDH1 previamente determinada foi analisada em relação aos níveis de expressões de mRNA do CXCR7, CXCR4 e HIF1alfa, combinado com os parâmetros clínico-patológicos e sobrevida global. Adicionalmente, a expressão proteica do CXCR7 foi analisada por imuno-histoquímica em astrocitomas de diferentes graus e em linhagem celular de glioma (U87MG) por microscopia confocal. Resultados: Houve diferença significativa nos níveis de expressão dos genes estudados entre astrocitomas e NN (p < 0,001). Na análise da expressão gênica associada nos AGII não se observou correlação entre os níveis de expressão de CXCR7/HIF1alfa (p = 0,548); observou-se correlação significativa entre CXCR7/IDH1 (p < 0,001) e CXCR7/CXCR4 (p = 0,042). Nos GBM houve correlação significativa entre CXCR7/CXCR4 (p = 0,002), CXCR7/IDH1 (p < 0,001) e CXCR7/HIF1alfa (p = 0,008). Hiperexpressão do HIF1alfa foi associado com maior expressão do CXCR7 e CXCR4 (p = 0,001), enquanto a presença de IDH1 mutado foi associada a menor expressão de mRNA do CXCR7 e CXCR4 (p = 0,009). A expressão proteica de CXCR7 foi identificada em todas as amostras estudadas, e aumentou com malignidade. A proteína CXCR7, na linha celular U87MG, foi localizada principalmente na membrana celular. Conclusão: O CXCR7 é um gene diferencialmente expresso em astrocitomas difusamente infiltrativos em relação tecido cerebral não neoplásico. O nível de expressão do CXCR7 correlacionou-se significativamente com os níveis de expressão do CXCR4 e IDH1 nos AGII e com CXCR4, IDH1 e HIF-1alfa nos GBM. O nível de expressão elevado do CXCR7 e CXCR4 correlacionou-se com nível elevado de expressão de HIF-1a, enquanto a presença da mutação do IDH1 associou-se a níveis reduzidos de CXCR7 e CXCR4. Não se observou associação significativa entre os níveis de expressão de CXCR7 e CXCR4 com os dados de sobrevida / Introduction: There is abundant evidence showing that chemokine CXCL12 and its receptor CXCR4 are involved in glioma progression and angiogenesis. CXCR4 is upregulated by HIF1alfa. The CXCR7, a recent additional receptor for CXCL12 with higher affinity than CXCR4 has raised key issues on glioma cell migration. The aim of this study is to investigate the CXCR7 mRNA expression in diffuse astrocytoma tissues and to evaluate its interactions with CXCR4 and HIF1alfa expression and IDH1 mutation. Methods: CXCR7, CXCR4, IDH1 and HIF1alfa expressions were evaluated by quantitative real-time PCR (qRT-PCR) in 129 frozen samples of astrocytoma (25 diffuse astrocytomas - AGII, 18 anaplastic astrocytomas - AGIII and 86 glioblastomas - GBM) and 22 samples of non-neoplastic tissue cerebral (NN) from epilepsy surgery. IDH1 mutation status was analyzed with CXCR7, CXCR4 e HIF1alfa mRNA expressions, matched with clinicopathological parameters and overall survival time. Furthermore, CXCR7 protein expression was analyzed by immunohistochemistry in different grades of astrocytoma and in glioma cell line (U87MG) by confocal microscopy. Results: There was significant difference in the expression levels of the genes studied between astrocytomas and NN (p < 0.001). The analysis of associated gene expressions in AGII showed no significant correlation between CXCR7/HIF1alfa (p = 0.548); there was significant correlation between CXCR7/CXCR4 (p = 0.042) and CXCR7/IDH1 (p = 0.008). In GBM, there were significant correlations between CXCR7/CXCR4 (p = 0.002), CXCR7/IDH1 (p < 0.001) and CXCR7/HIF1alfa (p = 0.008). HIF1alfa overexpression was associated with higher expressions of CXCR7 and CXCR4 (p = 0.001), while presence of IDH1 mutation was associated with lower CXCR7 and CXCR4 mRNA expressions (p = 0.009). Protein expression was identified in all samples studied, and it increased with malignancy. CXCR7 protein, in U87MG cell line, was mainly localized in the cellular membrane. Conclusion: CXCR7 was overexpressed in astrocytoma of different grades of malignancy compared to non-neoplastic brain tissue. CXCR7 expression levels correlates with CXCR4 and IDH1 in AGII and CXCR4, IDH1 and HIF1alfa in GBM. Overexpression HIF1alfa was related with higher expressions of CXCR7 and CXCR4, otherwise presence of IDH1 mutation related with lower expression of both genes. Protein expression level was associated with the degree of malignancy. The results revealed no significant association between CXCR7 and CXCR4 expression and survival data