Overexpression of bHLH domain of HIF-1 failed to inhibit the HIF-1 transcriptional activity in hypoxia

BACKGROUND: Hypoxia inducible factor-1 (HIF-1) is considered as the most activated transcriptional factor in response to low oxygen level or hypoxia. HIF-1 binds the hypoxia response element (HRE) sequence in the promoter of different genes, mainly through the bHLH domain and activates the transcription of genes, especially those involved in angiogenesis and EMT. Considering the critical role of bHLH in binding HIF-1 to the HRE sequence, we hypothesized that bHLH could be a promising candidate to be targeted in hypoxia condition. METHODS: We inserted an inhibitory bHLH (ibHLH) domain in a pIRES2-EGFP vector and transfected HEK293T cells with either the control vector or the designed construct. The ibHLH domain consisted of bHLH domains of both HIF-1a and Arnt, capable of competing with HIF-1 in binding to HRE sequences. The transfected cells were then treated with 200 µM of cobalt chloride (CoCl2) for 48 h to induce hypoxia. Real-time PCR and western blot were performed to evaluate the effect of ibHLH on the genes and proteins involved in angiogenesis and EMT. RESULTS: Hypoxia was successfully induced in the HEK293T cell line as the gene expression of VEGF, vimentin, and ß-catenin were significantly increased after treatment of untransfected HEK293T cells with 200 µM CoCl2. The gene expression of VEGF, vimentin, and ß-catenin and protein level of ß-catenin were significantly decreased in the cells transfected with either control or ibHLH vectors in hypoxia. However, ibHLH failed to be effective on these genes and the protein level of ß-catenin, when compared to the control vector. We also observed that overexpression of ibHLH had more inhibitory effect on gene and protein expression of N-cadherin compared to the control vector. However, it was not statistically significant. CONCLUSION: bHLH has been reported to be an important domain involved in the DNA binding activity of HIF. However, we found that targeting this domain is not sufficient to inhibit the endogenous HIF-1 transcriptional activity. Further studies about the function of critical domains of HIF-1 are necessary for developing a specific HIF-1 inhibitor.

Contexto sociodemográfico de los homicidios en México D.F.: un análisis espacial / Sociodemographic context of homicide in Mexico City: a spatial analysis

OBJETIVO:Investigar el patrón de distribución espacial de la tasa de homicidios y su relación con las características sociodemográficas en las delegaciones de Benito Juárez, Coyoacán y Cuauhtémoc de la Ciudad de México en el año 2010. MÉTODOS: Estudio inferencial de corte transversal que usa métodos de análisis espacial para estudiar la asociación espacial de la tasa de homicidios y las características demográficas. La asociación espacial fue determinada a través del cociente de localización, análisis de regresión múltiple y el uso de la regresión geográficamente ponderada. RESULTADOS: Los homicidios muestran un patrón de localización heterogéneo con altas tasas en zonas con uso del suelo no residencial, con baja densidad de población y baja marginación. CONCLUSIONES: El uso de herramientas de análisis espacial son instrumentos poderosos para el diseño de políticas de seguridad pública preventiva y recreativa que busquen reducir la mortalidad por causas externas como homicidios.

OBJECTIVE:Investigate the spatial distribution pattern of the homicide rate and its relation to sociodemographic features in the Benito Juárez, Coyoacán, and Cuauhtémoc districts of Mexico City in 2010. METHODS: Inferential cross-sectional study that uses spatial analysis methods to study the spatial association of the homicide rate and demographic features. Spatial association was determined through the location quotient, multiple regression analysis, and the use of geographically weighted regression. RESULTS: Homicides show a heterogeneous location pattern with high rates in areas with non-residential land use, low population density, and low marginalization. CONCLUSIONS: Spatial analysis tools are powerful instruments for the design of prevention- and recreation-focused public safety policies that aim to reduce mortality from external causes such as homicides.

mRNA expression profile of selected oxygen sensing genes in the lung during recovery from chronic hypoxia

This study analyzed the time dependence decay of the mRNA of selected genes important for the hypoxia response. The genes chosen were the two isoforms of hypoxia-inducible factors, the three isoforms of the prolyl hydroxylase domain protein, the vascular endothelial growth factor and endothelial nitric oxide synthase. mRNA and proteins were extracted from lungs obtained from control, hypoxic and 15 minutes normoxic recovered rats and analyzed by Real-time RT-PCR or by the Western Blot technique. Results indicated that in normoxia isoform 2á was the more represented hypoxia-inducible factor mRNA, and among the prolyl hydroxylase domain transcripts, isoform 3 was the least abundant. Moreover, in chronic hypoxia only hypoxia-inducible factor 1α and prolyl hydroxylase domain protein 3 increased significantly, while after 15 minutes of recovery all the mRNAs tested were decreased except endothelial nitric oxide synthase mRNA. In terms of proteins, hypoxia-inducible 1α was the isoform more significant in the nucleus, while 2á predominated in the cytosol. While the former was steady even after a brief recovery from hypoxia, the latter underwent a strong degradation. In conclusion we showed the relevance of the decay in the mRNA and protein levels upon re-oxygenation in normoxia. We believe that this has to be considered in research studies dealing with recovery from hypoxia.

Respuesta a la hipoxia: un mecanismo sistemico basado en el control de la expresion genica / Response to hypoxia: a systemic mechanism based on the control of gene expression

La respuesta hipóxica, sobre la que se dispone de nuevos datos críticamente importantes, puede esquematizarse en tres sistemas, vg. de detección o sensor de oxígeno, de regulación, que controla la expresión génica y efector. El elemento principal de organización del sistema regulador es un factor de transcripción específico, el factor inducible por hipoxia 1 (HIF-1). En presencia de oxígeno, la subunidad α del HIF-1 (HIF-1α) se modifica por las hidroxilasas, que constituyen el punto central del mecanismo sensor, induciendo su catabolismo por el proteosoma. Por el contrario, en hipoxia, o en presencia de algunos factores de crecimiento que incrementan su síntesis, el HIF-1α se transloca al núcleo, donde, unido al HIF-1β, actúa como factor transcripcional de genes con elementos de respuesta hipóxica (HRE) en su promotor. Estos regulan lasíntesis de una amplia serie de proteínas, que abarcan desde enzimas respiratorias y transportadores hasta hormonas involucradas en la regulación a escala del organismo de la circulación y la eritropoyesis. El papel del HIF-1 no se restringe a la mera inducción de una respuesta adaptativa a la falta de oxígeno, sino que participa significativamente en los mecanismos de reparación celular. Una simple lista de algunas alteraciones de importância fisiopatológica, tanto estimulatorias como inhibitorias, que involucran al sistema de HIF-1, incluiría: enfermedad pulmonar crónica, adaptación al tabaco/humo, anemia/hemorragia, isquemia/reperfusión, crecimiento, vascularización y resistencia celular de los tumores, preeclampsia y crecimiento intrauterino retardado, hiper o hipovascularización retiniana, sobredosis de fármacos, enfermedad inflamatoria intestinal y curación de heridas. Esta sola enumeración ilustra la importancia de este mecanismo. .

New, critically important data have been recently generated about the response to hypoxia. This response can be schematized in three main systems or functions, ie, detectional or oxygen sensing, regulatory, which controls gene expression and effector. The principal organizer of the regulatory branch is a specific transcription factor, the hypoxia-inducible factor 1 (HIF-1). In the presence of oxygen, the α subunit of HIF-1 (HIF-1α) is modified by hydroxylases, that represent the central point of the oxygen sensing mechanism. This type of hydroxylation induces HIF-1α catabolism by the proteosome. On the contrary, in hypoxia, or in the presence of certain growth factors that increase HIF-1α synthesis, HIF-1α translocates to the nucleus, where it binds HIF-1β, and thence acts on transcription of genes carrying hypoxia responsive elements (HRE) on their promoters. These genes regulate the synthesis of an ample series of proteins, which span from respiratory enzymes and transporters to hormones regulating circulation and erythropoiesis. The role of HIF-1α is not restricted to the mere induction of adaptation to decreased oxygen: instead, it significantly participates in cell repairing mechanisms. A simple list of some of the stimulatory or inhibitory alterations of pathophysiological importance involving the HIF-1 system, would include: chronic lung disease, smoking adaptation, anemia/hemorrhage, ischemia/reperfusion, growth, vascularization and cell resistance of tumors, preeclampsia and intrauterine growth retardation, retinal hyper ohypovascularization, drug intoxications, bowel inflammatory disease and wound repair. This list illustrates by itself the importance of the mechanism herein reviewed.

Las arterias pulmonares distales en los habitantes de las alturas / The pulmonary distal arteries at high altitude population

El estudio de las arterias pulmonares, en el hombre y en los animales que viven en las grandes alturas, ha demostrado la existencia de varios cambios morfológicos, como la muscularización de las arteriolas así como la proliferación de células musculares lisas en la íntima de las arterias distales. Estos cambios están relacionados con una vasoconstricción como respuesta a la hipoxia y al desarrollo de una hipertensión arterial pulmonar que, en algunos casos, se asocian con hipertrofia ventricular derecha e insuficiencia cardiaca congestiva, como en la enfermedad subaguda de la altura, descrita en el Himalaya y en los Andes en el hombre y en ciertas especies animales. Una posible pérdida de la respuesta vasoconstrictiva por transmisión genética a la hipoxia crónica se ha observado como una aclimatización o adaptación a las grandes alturas.