Research progress on the mechanism of phenotypic transformation of pulmonary artery smooth muscle cells induced by hypoxia / 浙江大学学报·医学版

Phenotypic transformation of pulmonary artery smooth muscle cells (PASMCs) is a key factor in pulmonary vascular remodeling. Inhibiting or reversing phenotypic transformation can inhibit pulmonary vascular remodeling and control the progression of hypoxic pulmonary hypertension. Recent studies have shown that hypoxia causes intracellular peroxide metabolism to induce oxidative stress, induces multi-pathway signal transduction, including those related to autophagy, endoplasmic reticulum stress and mitochondrial dysfunction, and also induces non-coding RNA regulation of cell marker protein expression, resulting in PASMCs phenotypic transformation. This article reviews recent research progress on mechanisms of hypoxia-induced phenotypic transformation of PASMCs, which may be helpful for finding targets to inhibit phenotypic transformation and to improve pulmonary vascular remodeling diseases such as hypoxia-induced pulmonary hypertension.

Activation of HIF-1α/ACLY signaling axis promotes progression of clear cell renal cell carcinoma with VHL inactivation mutation / 中华病理学杂志

Objective: To explore the potential pathogenesis of clear cell renal cell carcinoma (ccRCC) based on the HIF-1α/ACLY signaling pathway, as well as to provide new ideas for the treatment of ccRCC. Methods: Seventy-eight ccRCC cases diagnosed at the First Affiliated Hospital of Soochow University, Suzhou, China were collected. The VHL mutation was examined using exon sequencing. The expression of HIF-1α/ACLY in VHL-mutated ccRCC was evaluated using immunohistochemical staining and further validated in VHL-mutated ccRCC cell lines (786-O, A498, UM-RC-2, SNU-333, and Caki-2) using Western blot. The mRNA and protein levels of ACLY were detected using real-time quantitative PCR and Western blot after overexpression or interference with HIF-1α in ccRCC cell lines. HeLa cells were treated with CoCl2 and hypoxia (1%O2) to activate HIF-1α and then subject to the detection of the ACLY mRNA and protein levels. The potential molecular mechanism of HIF-1α-induced ACLY activation was explored through JASPAR database combined with chromatin immunoprecipitation assay (ChIP) and luciferase reporter gene assay. The effect of HIF-1α/ACLY regulation axis on lipid accumulation was detected using BODIPY staining and other cell biological techniques. The expression of ACLY was compared between patients with ccRCC and those with benign lesions, and the feasibility of ACLY as a prognostic indicator for ccRCC was explored through survival analysis. Results: Exon sequencing revealed that 55 (70.5%) of the 78 ccRCC patients harbored a VHL inactivation mutation, and HIF-1α expression was associated with ACLY protein levels. The protein levels of ACLY and HIF-1α in ccRCC cell lines carrying VHL mutation were also correlated to various degrees. Overexpression of HIF-1α in A498 cells increased the mRNA and protein levels of ACLY, and knockdown of HIF-1α in Caki-2 cells inhibited the mRNA and protein levels of ACLY (P<0.001 for all). CoCl2 and hypoxia treatment significantly increased the mRNA and protein levels of ACLY by activating HIF-1α (P<0.001 for all). The quantification of transcriptional activity of luciferase reporter gene and ChIP-qPCR results suggested that HIF-1α could directly bind to ACLY promoter region to transcriptionally activate ACLY expression and increase ACLY protein level (P<0.001 for all). The results of BODIPY staining suggested that the content of free fatty acids in cell lines was associated with the levels of HIF-1α and ACLY. The depletion of HIF-1α could effectively reduce the accumulation of lipid in cells, while the overexpression of ACLY could reverse this process. At the same time, cell function experiments showed that the proliferation rate of ccRCC cells with HIF-1α knockdown was significantly decreased, and overexpression of ACLY could restore proliferation of these tumor cells (P<0.001). Survival analysis further showed that compared with the ccRCC patients with low ACLY expression, the ccRCC patients with high ACLY expression had a poorer prognosis and a shorter median survival (P<0.001). Conclusions: VHL mutation-mediated HIF-1α overexpression in ccRCC promotes lipid synthesis and tumor progression by activating ACLY. Targeting the HIF-1α/ACLY signaling axis may provide a theoretical basis for the clinical diagnosis and treatment of ccRCC.

Down-regulation of miR-488 targeting to promote Jag1 expression inhibits hypoxia-reoxygenation myocardial H9c2 cell damage / 中华医学遗传学杂志

OBJECTIVE@#To study the effect of down-regulating miR-488 targeting Jag1 on the injury of hypoxia-reoxygenation myocardial H9c2 cells.@*METHODS@#A hypoxic-reoxygenated myocardial H9c2 cell injury model was constructed. miR-488 inhibitor was used to transfect the cells. CCK-8 method and flow cytometry were used to detect cell proliferation and apoptosis in each group. Lactate dehydrogenase (LDH), superoxide dismutase (SOD), malonaldehyde (MDA), catalase (CAT) levels were detected. Western blotting was used to detect the expression of Bcl-2 associated X Protein (Bax) and B cell lymphoma/lewkmia-2 (Bcl-2). Target genes of miR-488 were predicted, and a luciferase reporter system was used to verify the targeting relationship between the two. Myocardial H9c2 cells were co-transfected with miR-488 inhibitor and Jag1 siRNA, and treated with hypoxia and reoxygenation, cell proliferation, apoptosis, LDH, SOD, MDA, CAT levels, and Bax, Bcl-2 protein expression were detected.@*RESULTS@#The expression of miR-488 in the hypoxia-reoxygenated myocardial H9c2 cells was increased, along with reduced cell proliferation, increased apoptosis, increased Bax protein expression, decreased Bcl-2 protein expression, increased MDA, decreased CAT and SOD, and increased LDH level in the supernatant of cell culture. When myocardial H9c2 cells were transfected with miR-488 inhibitor and treated with hypoxia and reoxygenation, the expression of miR-488 was decreased, along with increased cell proliferation, decreased apoptosis, decreased Bax protein expression, increased Bcl-2 protein expression, decreased MDA, increased CAT and SOD, and decreased LDH level in the supernatant of cell culture. Down-regulation of miR-488 could target and down-regulate Jag1 expression. And Jag1 siRNA could reverse the effect of miR-488 inhibitor on the proliferation, apoptosis, LDH, SOD, MDA, CAT levels and the expression of Bax and Bcl-2 of hypoxic-reoxygenated myocardial H9c2 cells.@*CONCLUSION@#Down-regulating miR-488 targeted Jag1 can attenuate hypoxia-reoxygenation induced myocardial H9c2 cell injury.

Transcriptional Profile Alteration of Peripheral Blood in Chronic Hypoxia / 中国医学科学杂志(英文版)

Objective Many physiological and pathological conditions, including cyanotic congenital heart diseases (CCHD), are accompanied by chronic hypoxia, which might interfere with the transcription process. However, the transcriptome profile in peripheral blood under hypoxia is still unidentified. The present work aimed to explore the transcriptional profile alteration of peripheral blood in chronic hypoxia. Methods The present study used a chronic hypoxia rat model to simulate the hypoxic state of CCHD patients. Two groups of Sprague-Dawley rats (n=6 per group) were either exposed to hypoxia (10% O2) or normoxia (21% O2) for 3 weeks. Body weight was measured weekly. Peripheral blood was collected and total RNA was extracted for RNA-Seq at the end of the hypoxia treatment. After quality assessment, the library was sequenced by the Illumina Hiseq platform. The differentially expressed genes were screened (false discovery rate<0.05 and fold change>2). The functional annotation analysis and cluster analysis of differentially expressed genes were performed based on the adjusted P-value (padj<0.05). Results Compared with the control group, the body weight of the rats in the hypoxia group was significantly lowered (P<0.01). RNA-Seq results showed that the transcriptome patterns of the two groups had significant differences. In total, 872 genes were identified as differentially expressed. Among all, 803 genes were down-regulated, while only 69 genes were up-regulated in the hypoxia group. The functional enrichment analysis of the 872 genes showed that multiple biological processes involved, such as porphyrin-containing compound metabolic process, hemoglobin complex and oxygen transporter activity. Conclusions Our study demonstrated the transcriptional profile alteration in peripheral blood of chronic hypoxia rat model. This study provided basic data and directions to further understand the physiological and pathological changes in patients with CCHD.

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.

Hypoxia Induces Epithelial-Mesenchymal Transition in Follicular Thyroid Cancer: Involvement of Regulation of Twist by Hypoxia Inducible Factor-1alpha

PURPOSE: Although follicular thyroid cancer (FTC) has a relatively fair prognosis, distant metastasis sometimes results in poor prognosis and survival. There is little understanding of the mechanisms contributing to the aggressiveness potential of thyroid cancer. We showed that hypoxia inducible factor-1alpha (HIF-1alpha) induced aggressiveness in FTC cells and identified the underlying mechanism of the HIF-1alpha-induced invasive characteristics. MATERIALS AND METHODS: Cells were cultured under controlled hypoxic environments (1% O2) or normoxic conditions. The effect of hypoxia on HIF-1alpha, and epithelial-to-mesenchymal transition (EMT) related markers were evaluated by quantitative real-time PCR, Western blot analysis and immunocytochemistry. Invasion and wound healing assay were conducted to identify functional character of EMT. The involvement of HIF-1alpha and Twist in EMT were studied using gene overexpression or silencing. After orthotopic nude mouse model was established using the cells transfected with lentiviral shHIF-1alpha, tissue analysis was done. RESULTS: Hypoxia induces HIF-1alpha expression and EMT, including typical morphologic changes, cadherin shift, and increased vimentin expression. We showed that overexpression of HIF-1alpha via transfection resulted in the aforementioned changes without hypoxia, and repression of HIF-1alpha with RNA interference suppressed hypoxia-induced HIF-1alpha and EMT. Furthermore, we also observed that Twist expression was regulated by HIF-1alpha. These were confirmed in the orthotopic FTC model. CONCLUSION: Hypoxia induced HIF-1alpha, which in turn induced EMT, resulting in the increased capacity for invasion and migration of cells via regulation of the Twist signal pathway in FTC cells. These findings provide insight into a possible therapeutic strategy to prevent invasive and metastatic FTC.

Hypobaric hypoxia-mediated protein expression in plasma of susceptible & tolerant rats.

Background & objectives: Low availability of oxygen at high altitudes has a great impact on the human life processes. There is a widespread interest and need to find out protein(s) that are possibly involved in mediating tolerance to hypobaric hypoxia. We undertook this study to identify and characterize protein expression in plasma of hypoxia susceptible and tolerant rats. Methods: Male albino Sprague Dawley rats were segregated into susceptible and tolerant groups on the basis of their gasping time when exposed to simulated hypobaric hypoxia of 32,000 ft (9,754 m) at 32ºC. Comparative proteome profiling of blood plasma of hypoxia susceptible and tolerant individuals was performed using 2-dimentional (2-D) gel electrophoresis. Results: Three proteins with higher expression levels were selected separately from tolerant and susceptible samples. Characterization of these proteins from tolerant sample using MALDI-TOF/TOF and MASCOT search indicated their homology with two different super-families viz. NADB-Rossmann superfamily (Rab GDP dissociation inhibitor β) and Transferrin superfamily (two Serotransferrins), having potential role in imparting tolerance against hypoxia. Three high level upregulated proteins were characterized from blood plasma of hypoxia susceptible animals showing similarity with threonine tRNA ligase (mitochondrial), carbohydrate sulphotransferase 7 and aspartate tRNA ligase (cytoplasmic) that play a role in ATP binding, carbohydrate metabolism and protein biosynthesis, respectively. Interpretation & conclusions: Our results indicated that rats segregated into hypoxia sensitive and tolerant based on their gasping time showed differential expression of proteins in blood plasma. Characterization of these differentially expressed proteins will lead to better understanding of molecular responses occurring during hypoxia and subsequently development of biomarkers for categorization of hypoxia susceptible and tolerant individuals.

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.

Hypoxia and angiogenesis: regulation of hypoxia-inducible factors via novel binding factors

The mechanisms that regulate angiogenesis in hypoxia or hypoxic microenvironment are modulated by several pro- and antiangiogenic factors. Hypoxia-inducible factors (HIFs) have been established as the basic and major inducers of angiogenesis, but understanding the role of interacting proteins is becoming increasingly important to elucidate the angiogenic processes of a hypoxic response. In particular, with regard to wound healing and the novel therapies for vascular disorders such as ischemic brain and heart attack, it is essential to gain insights in the formation and regulation of HIF transcriptional machineries related to angiogenesis. Further, identification of alternative ways of inhibiting tumor growth by disrupting the growth-triggering mechanisms of increasing vascular supply via angiogenesis depends on the knowledge of how tumor cells develop their own vasculature. Here, we review our findings on the interactions of basic HIFs, HIF-1alpha and HIF-2alpha, with their regulatory binding proteins, histone deacetylase 7 (HDAC7) and translation initiation factor 6 (Int6), respectively. The present results and discussion revealed new regulatory interactions of HIF-related mechanisms.

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.

Hypoxia activates signal transducers and activators of transcription 5 (STAT5) and increases its binding activity to the GAS element in mammary epithelial cells

STATs (signal transducers and activators of transcription) are proteins with dual functions: signal transducers in the cytoplasm and transcriptional activators in the nucleus. STAT proteins act as transcription factors activated by phosphorylation on its tyrosine residues upon stimulation by various cytokines. The phosphorylated STAT molecules then form homo- or heterodimers through SH2-mediated interaction and translocate into the nucleus to activate the transcription of various target genes. STAT5 recognizes the interferon-gamma activated site TTCNNNGAA (GAS sequence) in the promoter region of the beta-casein gene. Except for prolactin-dependent beta-casein production in mammary gland cells, the biological consequences of STAT5a activation in various systems are not clear. Here we showed that STAT5a was phosphorylated 10 min after desferrioxamine (DFO) treatment, and reached a maximum induction at 4 h in mammary epithelial cells (HC11) and transfected COS-7 cells. Under hypoxic conditions (2% O2), a maximal phosphorylation of STAT5a was observed within 6 h. EMSA (electrophoretic mobility shift assay) showed that DFO or hypoxia enhanced the binding activities of STAT5a DNA to beta-casein gene promoter in mammary epithelial cells (HC11) and transfected COS-7 cells. These results showed that DFO or hypoxia induces tyrosine phosphorylation of STAT5a and also increases the binding activity of STAT5a DNA in mammary epithelial cells. Our data suggest that the STAT5 may act as a mediator in hypoxia-mediated gene expression.

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.