Impact of Extrinsic and Intrinsic Hypoxia on Catecholamine Biosynthesis in Absence or Presence of Hif2α in Pheochromocytoma Cells

Pheochromocytomas and paragangliomas (PPGLs) with activated pseudohypoxic pathways are associated with an immature catecholamine phenotype and carry a higher risk for metastasis. For improved understanding of the underlying mechanisms we investigated the impact of hypoxia and pseudohypoxia on catecholamine biosynthesis in pheochromocytoma cells naturally lacking Hif2α (MPC and MTT) or expressing both Hif1α and Hif2α (PC12). Cultivation under extrinsic hypoxia or in spheroid culture (intrinsic hypoxia) increased cellular dopamine and norepinephrine contents in all cell lines. To distinguish further between Hif1α- and Hif2α-driven effects we expressed Hif2α in MTT and MPC-mCherry cells (naturally lacking Hif2α). Presence of Hif2α resulted in similarly increased cellular dopamine and norepinephrine under hypoxia as in the control cells. Furthermore, hypoxia resulted in enhanced phosphorylation of tyrosine hydroxylase (TH). A specific knockdown of Hif1α in PC12 diminished these effects. Pseudohypoxic conditions, simulated by expression of Hif2α under normoxia resulted in increased TH phosphorylation, further stimulated by extrinsic hypoxia. Correlations with PPGL tissue data led us to conclude that catecholamine biosynthesis under hypoxia is mainly mediated through increased phosphorylation of TH, regulated as a short-term response (24-48 h) by HIF1α. Continuous activation of hypoxia-related genes under pseudohypoxia leads to a HIF2α-mediated phosphorylation of TH (permanent status).Funding: This research was funded by the Deutsche Forschungsgemeinschaft (DFG) within the CRC/Transregio205/1 (project number: 314061271-TRR 205), Project No. B12 (N.B. and G.E.), Project No. B10 (S.R., J.P. and M.U.)and Project No. S01 (A.W., C.G. and M.P.) “The Adrenal: Central Relay in Health and Disease“, and by theParadi erence Foundation (N.B., I.P., S.R. and G.E.).S

Genotypes at the APOE and SCA2 loci do not predict the course of multiple sclerosis in patients of Portuguese origin

Prova tipográfica (In Press)Multiple sclerosis (MS) is a demyelinating disease that affects about one in 500 young Europeans. In order to test the previously proposed influence of the APOE and SCA2 loci on susceptibility to MS, we studied these loci in 243 Portuguese patients and 192 healthy controls and both parents of 92 patients. We did not detect any significant difference when APOE and SCA2 allele frequencies of cases and controls were compared, or when we compared cases with different forms of the disease. Disequilibrium of transmission was tested for both loci in the 92 trios, and we did not observe segregation distortion. To test the influence of the APOE o4 and SCA2 22 CAGs alleles on severity of disease, we compared age at onset and progression rate between groups with and without those alleles. We did not observe an association of the o4 or the 22 CAGs alleles with rate of progression in our total patient population; allele o4 was associated with increased rate of progression of MS in a subset of patients with less than 10 years of the disease. However, globally in the Portuguese population, the APOE and SCA2 genes do not seem to be useful in the clinical context as prognostic markers of this disorder.Fundação para a Ciência e a Tecnologia (FCT) - grant SFRH/BD/9111/2002.Serono Portugal

Treatment of Pheochromocytoma Cells with Recurrent Cycles of Hypoxia: A New Pseudohypoxic In Vitro Model

Continuous activation of hypoxia pathways in pheochromocytomas and paragangliomas (PPGLs) is associated with higher disease aggressiveness, for which effective treatment strategies are still missing. Most of the commonly used in vitro models lack characteristics of these pseudohypoxic tumors, including elevated expression of hypoxia-inducible factor (HIF) 2α. To address this shortcoming, we investigated whether recurrent hypoxia cycles lead to continuous activation of hypoxia pathways under normoxic conditions and whether this pseudohypoxia is associated with increased cellular aggressiveness. Rat pheochromocytoma cells (PC12) were incubated under hypoxia for 24 h every 3-4 days, up to 20 hypoxia-reoxygenation cycles, resulting in PC12 Z20 cells. PC12 Z20 control cells were obtained by synchronous cultivation under normoxia. RNA sequencing revealed upregulation of HIF2α in PC12 Z20 cells and a pseudohypoxic gene signature that overlapped with the gene signature of pseudohypoxic PPGLs. PC12 Z20 cells showed a higher growth rate, and the migration and adhesion capacity were significantly increased compared with control cells. Changes in global methylation, together with the pseudohypoxic conditions, may be responsible for the increased aggressiveness of this new model. The established sub-cell line with characteristics of pseudohypoxic PPGLs represent a complementary model for further investigations, for example, with regard to new therapeutic approaches. Keywords: drug resistance; epigenetic; hypermethylation; hypoxia resistance; metastasis; paraganglioma; pheochromocytoma; pseudohypoxia

O desafio de controlar o tabagismo em um hospital universitário

Objetivo: Identifi car as ações desenvolvidas por uma Comissão de Controle do Tabagismo (CCT) para controlar o fumo do ambiente hospitalar. Métodos: Pesquisa documental descritivo-exploratória retrospectiva, realizada em hospital universitário do sul do Brasil, em 2014. Construiu-se um banco de dados com o conteúdo das atas de reuniões da CCT e relatório das rondas realizadas, o qual foi analisado de forma descritiva. Buscou-se identifi car as ações mais relevantes no período de 2005 a 2014. Resultados: A CCT implementou o Programa Ambiente Livre do Tabaco, restringiu o consumo do cigarro aos fumódromos e posteriormente desativou os mesmos. Permaneceu somente uma área externa de tolerância ao fumo até 2014, a qual foi desativada. Conclusões: As ações da CCT contribuíram para controlar o fumo do ambiente hospitalar. Espera-se que este estudo sirva de incentivo e modelo para outras instituições.Objective: To identify the actions taken by the Commission of Tobacco Control (CTC) to control smoking in the hospital environment. Methods: Descriptive and exploratory retrospective documentary research conducted at a university hospital in southern Brazil, in 2014. The content of the minutes of CTC meetings was used to create a database, and the rounds reports were descriptively analyzed. We sought to identify the most relevant actions from 2005 to 2014. Results: The CTC implemented the Tobacco-Free Environment programme restricted cigarette smoking to designated areas and subsequently deactivated these areas. The only remaining outdoor smoking area in 2014 was deactivated. Conclusion: CTC actions have contributed to tobacco control in the hospital environment. This study will hopefully serve as a model to encourage other institutions to implement similar actions.Objetivo: Identifi car las medidas adoptadas por la Comisión de Control del Tabaco (CCT) para controlar el tabaquismo del entorno hospitalario. Métodos: Investigación documental retrospectiva descriptiva y exploratoria, realizada en hospital universitario en el sur de Brasil, en 2014. Construyó una base de datos con el contenido de las actas de las reuniones del CCT y la notifi cación de las rondas realizadas, que se analizó descriptivamente. Se buscó identifi car las acciones más relevantes, en el periodo 2005-2014. Resultados: El CCT implementó el Programa de Ambiente Libre de Tabaco, restringió el consumo de cigarrillos a las instalaciones designadas, y luego los convirtió. Sólo quedaba un área externa de la tolerancia al consumo de tabaco en 2014, que fue desactivado. Conclusiones: Las acciones del CCT ayudaron a controlar el tabaquismo del hospital. Se espera que este estudio sirva de estímulo y modelo para otras instituciones

O desafio de controlar o tabagismo em um hospital universitário

Objetivo: Identifi car as ações desenvolvidas por uma Comissão de Controle do Tabagismo (CCT) para controlar o fumo do ambiente hospitalar. Métodos: Pesquisa documental descritivo-exploratória retrospectiva, realizada em hospital universitário do sul do Brasil, em 2014. Construiu-se um banco de dados com o conteúdo das atas de reuniões da CCT e relatório das rondas realizadas, o qual foi analisado de forma descritiva. Buscou-se identifi car as ações mais relevantes no período de 2005 a 2014. Resultados: A CCT implementou o Programa Ambiente Livre do Tabaco, restringiu o consumo do cigarro aos fumódromos e posteriormente desativou os mesmos. Permaneceu somente uma área externa de tolerância ao fumo até 2014, a qual foi desativada. Conclusões: As ações da CCT contribuíram para controlar o fumo do ambiente hospitalar. Espera-se que este estudo sirva de incentivo e modelo para outras instituições.Objective: To identify the actions taken by the Commission of Tobacco Control (CTC) to control smoking in the hospital environment. Methods: Descriptive and exploratory retrospective documentary research conducted at a university hospital in southern Brazil, in 2014. The content of the minutes of CTC meetings was used to create a database, and the rounds reports were descriptively analyzed. We sought to identify the most relevant actions from 2005 to 2014. Results: The CTC implemented the Tobacco-Free Environment programme restricted cigarette smoking to designated areas and subsequently deactivated these areas. The only remaining outdoor smoking area in 2014 was deactivated. Conclusion: CTC actions have contributed to tobacco control in the hospital environment. This study will hopefully serve as a model to encourage other institutions to implement similar actions.Objetivo: Identifi car las medidas adoptadas por la Comisión de Control del Tabaco (CCT) para controlar el tabaquismo del entorno hospitalario. Métodos: Investigación documental retrospectiva descriptiva y exploratoria, realizada en hospital universitario en el sur de Brasil, en 2014. Construyó una base de datos con el contenido de las actas de las reuniones del CCT y la notifi cación de las rondas realizadas, que se analizó descriptivamente. Se buscó identifi car las acciones más relevantes, en el periodo 2005-2014. Resultados: El CCT implementó el Programa de Ambiente Libre de Tabaco, restringió el consumo de cigarrillos a las instalaciones designadas, y luego los convirtió. Sólo quedaba un área externa de la tolerancia al consumo de tabaco en 2014, que fue desactivado. Conclusiones: Las acciones del CCT ayudaron a controlar el tabaquismo del hospital. Se espera que este estudio sirva de estímulo y modelo para otras instituciones

The challenge of tobacco control at a university hospital

Objective: To identify the actions taken by the Commission of Tobacco Control (CTC) to control smoking in the hospital environment.Methods: Descriptive and exploratory retrospective documentary research conducted at a university hospital in southern Brazil, in 2014. The content of the minutes of CTC meetings was used to create a database, and the rounds reports were descriptively analyzed. We sought to identify the most relevant actions from 2005 to 2014.Results: The CTC implemented the Tobacco-Free Environment programme restricted cigarette smoking to designated areas and subsequently deactivated these areas. The only remaining outdoor smoking area in 2014 was deactivated.Conclusion: CTC actions have contributed to tobacco control in the hospital environment. This study will hopefully serve as a model to encourage other institutions to implement similar actions