Copper-dependent activation of hypoxia-inducible factor (HIF)-1: implications for ceruloplasmin regulation

Cellular oxygen partial pressure is sensed by a family of prolyl-4-hydroxylase domain (PHD) enzymes that modify hypoxia-inducible factor (HIF)alpha subunits. Upon hydroxylation under normoxic conditions, HIFalpha is bound by the von Hippel-Lindau tumor suppressor protein and targeted for proteasomal destruction. Since PHD activity is dependent on oxygen and ferrous iron, HIF-1 mediates not only oxygen- but also iron-regulated transcriptional gene expression. Here we show that copper (CuCl(2)) stabilizes nuclear HIF-1alpha under normoxic conditions, resulting in hypoxia-response element (HRE)-dependent reporter gene expression. In in vitro hydroxylation assays CuCl(2) inhibited prolyl-4-hydroxylation independently of the iron concentration. Ceruloplasmin, the main copper transport protein in the plasma and a known HIF-1 target in vitro, was also induced in vivo in the liver of hypoxic mice. Both hypoxia and CuCl(2) increased ceruloplasmin (as well as vascular endothelial growth factor [VEGF] and glucose transporter 1 [Glut-1]) mRNA levels in hepatoma cells, which was due to transcriptional induction of the ceruloplasmin gene (CP) promoter. In conclusion, our data suggest that PHD/HIF/HRE-dependent gene regulation can serve as a sensory system not only for oxygen and iron but also for copper metabolism, regulating the oxygen-, iron- and copper-binding transport proteins hemoglobin, transferrin, and ceruloplasmin, respectively

Time-Action Analysis (TAA) of the Surgical Technique Implanting the Collum Femoris Preserving (CFP) Hip Arthroplasty. TAASTIC trial Identifying pitfalls during the learning curve of surgeons participating in a subsequent randomized controlled trial (An observational study)

<p>Abstract</p> <p>Background</p> <p>Two types of methods are used to assess learning curves: outcome assessment and process assessment. Outcome measures are usually dichotomous rare events like complication rates and survival or require an extensive follow-up and are therefore often inadequate to monitor individual learning curves. Time-action analysis (TAA) is a tool to objectively determine the level of efficiency of individual steps of a surgical procedure.</p> <p>Methods/Design</p> <p>We are currently using TAA to determine the number of cases needed for surgeons to reach proficiency with a new innovative hip implant prior to initiating a multicentre RCT. By analysing the unedited video recordings of the first 20 procedures of each surgeon the number and duration of the actions needed for a surgeon to achieve his goal and the efficiency of these actions is measured. We constructed a taxonomy or list of actions which together describe the complete surgical procedure. In the taxonomy we categorised the procedure in 5 different Goal Oriented Phases (GOP):</p> <p>1. the incision phase</p> <p>2. the femoral phase</p> <p>3. the acetabulum phase</p> <p>4. the stem phase</p> <p>5. the closure pase</p> <p>Each GOP was subdivided in Goal Oriented Actions (GOA) and each GOA is subdivided in Separate Actions (SA) thereby defining all the necessary actions to complete the procedure. We grouped the SAs into GOAs since it would not be feasible to measure each SA. Using the video recordings, the duration of each GOA was recorded as well as the amount of delay. Delay consists of repetitions, waiting and additional actions. The nett GOA time is the total GOA time – delay and is a representation of the level of difficulty of each procedure. Efficiency is the percentage of nett GOA time during each procedure.</p> <p>Discussion</p> <p>This allows the construction of individual learning curves, assessment of the final skill level for each surgeon and comparison of different surgeons prior to participation in an RCT. We believe an objective and comparable assessment of skill level by process assessment can improve the value of a surgical RCT in situations where a learning curve is expected.</p

VEGF is upregulated by hypoxia-induced mitogenic factor via the PI-3K/Akt-NF-κB signaling pathway

BACKGROUND: Hypoxia-induced mitogenic factor (HIMF) is developmentally regulated and plays an important role in lung pathogenesis. We initially found that HIMF promotes vascular tubule formation in a matrigel plug model. In this study, we investigated the mechanisms which HIMF enhances expression of vascular endothelial growth factor (VEGF) in lung tissues and epithelial cells. METHODS: Recombinant HIMF protein was intratracheally instilled into adult mouse lungs, VEGF expression was examined by immunohistochemical staining and Western blot. The promoter-luciferase reporter assay, RT-PCR, and Western blot were performed to examine the effects of HIMF on VEGF expression in mouse lung epithelial cell line MLE-12. The activation of NF-kappa B (NF-κB) and phosphorylation of Akt, IKK and IκBα were examined by luciferase assay and Western blot, respectively. RESULTS: Intratracheal instillation of HIMF protein resulted in significant increase of VEGF, mainly localized to airway epithelial and alveolar type II cells. Deletion of NF-κB binding sites within VEGF promoter abolished HIMF-induced VEGF expression in MLE-12 cells, suggesting that activation of NF-κB is essential for VEGF upregulation induced by HIMF. Stimulation of lung epithelial cells by HIMF resulted in phosphorylation of IKK and IκBα, leading to activation of NF-κB. In addition, HIMF strongly induced Akt phosphorylation, and suppression of Akt activation by specific inhibitors and dominant negative mutants for PI-3K, and IKK or IκBα blocked HIMF-induced NF-κB activation and attenuated HIMF-induced VEGF production. CONCLUSION: These results suggest that HIMF enhances VEGF production in mouse lung epithelial cells in a PI-3K/Akt-NF-κB signaling pathway-dependent manner, and may play critical roles in pulmonary angiogenesis

In Vivo Isolation and Characterization of Stem Cells with Diverse Phenotypes Using Growth Factor Impregnated Biomatrices

BACKGROUND: The stimulation to differentiate into specific cell types for somatic stem cells is largely due to a series of internal and external signals coming from the microenvironment that surrounds the stem cell. Even though intensive research has been made, the basic mechanisms of plasticity and/or the molecules regulating stem cells proliferation and differentiation are not completely determined. Potential answers concerning the problems could be derived from the studies of stem cells in culture. METHODOLOGY/PRINCIPLE FINDINGS: We combine a new procedure (using the matrigel biopolymer supplemented with a selected cytokine/growth factor) with classic techniques such as light, confocal and electron microscopy, immunohistochemistry and cell culture, to perform an analysis on stem cells involved in the leech (Hirudo medicinalis) repair tissues. The leech has a relative anatomical simplicity and is a reliable model for studying a variety of basic events, such as tissue repair, which has a striking similarity with vertebrate responses. Our data demonstrate that the injection of an appropriate combination of the matrigel biopolymer supplemented with a selected cytokine/growth factor in the leech Hirudo medicinalis is a remarkably effective tool for isolating a specific cell population in vivo. A comparative analysis of biopolymer in vivo sorted stem cells indicates that VEGF recruited cells of a hematopoietic/endothelial phenotype whereas MCP-1/CCL2 isolated cells that were of an early myeloid lineage. CONCLUSION: Our paper describes, for the first time, a method allowing not only the isolation of a specific cell population in relation to the cytokine utilized but also the possibility to culture a precise cell type whose isolation is otherwise quite difficult. This approach could be broadly applied to isolate stem cells of diverse origins based on the recruitment stimuli employed

Association of Genetic Variants With Primary Open-Angle Glaucoma Among Individuals With African Ancestry.

Importance:Primary open-angle glaucoma presents with increased prevalence and a higher degree of clinical severity in populations of African ancestry compared with European or Asian ancestry. Despite this, individuals of African ancestry remain understudied in genomic research for blinding disorders. Objectives:To perform a genome-wide association study (GWAS) of African ancestry populations and evaluate potential mechanisms of pathogenesis for loci associated with primary open-angle glaucoma. Design, Settings, and Participants:A 2-stage GWAS with a discovery data set of 2320 individuals with primary open-angle glaucoma and 2121 control individuals without primary open-angle glaucoma. The validation stage included an additional 6937 affected individuals and 14 917 unaffected individuals using multicenter clinic- and population-based participant recruitment approaches. Study participants were recruited from Ghana, Nigeria, South Africa, the United States, Tanzania, Britain, Cameroon, Saudi Arabia, Brazil, the Democratic Republic of the Congo, Morocco, Peru, and Mali from 2003 to 2018. Individuals with primary open-angle glaucoma had open iridocorneal angles and displayed glaucomatous optic neuropathy with visual field defects. Elevated intraocular pressure was not included in the case definition. Control individuals had no elevated intraocular pressure and no signs of glaucoma. Exposures:Genetic variants associated with primary open-angle glaucoma. Main Outcomes and Measures:Presence of primary open-angle glaucoma. Genome-wide significance was defined as P &lt; 5 × 10-8 in the discovery stage and in the meta-analysis of combined discovery and validation data. Results:A total of 2320 individuals with primary open-angle glaucoma (mean [interquartile range] age, 64.6 [56-74] years; 1055 [45.5%] women) and 2121 individuals without primary open-angle glaucoma (mean [interquartile range] age, 63.4 [55-71] years; 1025 [48.3%] women) were included in the discovery GWAS. The GWAS discovery meta-analysis demonstrated association of variants at amyloid-β A4 precursor protein-binding family B member 2 (APBB2; chromosome 4, rs59892895T&gt;C) with primary open-angle glaucoma (odds ratio [OR], 1.32 [95% CI, 1.20-1.46]; P = 2 × 10-8). The association was validated in an analysis of an additional 6937 affected individuals and 14 917 unaffected individuals (OR, 1.15 [95% CI, 1.09-1.21]; P &lt; .001). Each copy of the rs59892895*C risk allele was associated with increased risk of primary open-angle glaucoma when all data were included in a meta-analysis (OR, 1.19 [95% CI, 1.14-1.25]; P = 4 × 10-13). The rs59892895*C risk allele was present at appreciable frequency only in African ancestry populations. In contrast, the rs59892895*C risk allele had a frequency of less than 0.1% in individuals of European or Asian ancestry. Conclusions and Relevance:In this genome-wide association study, variants at the APBB2 locus demonstrated differential association with primary open-angle glaucoma by ancestry. If validated in additional populations this finding may have implications for risk assessment and therapeutic strategies

The Noise Exposure Structured Interview (NESI): an instrument for the comprehensive estimation of lifetime noise exposure

Lifetime noise exposure is generally quantified by self report. The accuracy of retrospective self report is limited by respondent recall, but is also bound to be influenced by reporting procedures. Such procedures are of variable quality in current measures of lifetime noise exposure, and off-the-shelf instruments are not readily available. The Noise Exposure Structured Interview (NESI) represents an attempt to draw together some of the stronger elements of existing procedures and to provide solutions to their outstanding limitations. Reporting is not restricted to pre-specified exposure activities, and instead encompasses all activities that the respondent has experienced as noisy (defined based on sound level estimated from vocal effort). Changing exposure habits over time are reported by dividing the lifespan into discrete periods in which exposure habits were approximately stable, with life milestones used to aid recall. Exposure duration, sound level, and use of hearing protection are reported for each life period separately. Simple-to-follow methods are provided for the estimation of free-field sound level, the sound level emitted by personal listening devices, and the attenuation provided by hearing protective equipment. An energy-based means of combining the resulting data is supplied, along with a primarily energy-based method for incorporating firearm-noise exposure. Finally, the NESI acknowledges the need of some users to tailor the procedures; this flexibility is afforded and reasonable modifications are described. Competency needs of new users are addressed through detailed interview instructions (including troubleshooting tips) and a demonstration video. Limited evaluation data are available and future efforts at evaluation are proposed

Vascular regrowth following photodynamic therapy in the chicken embryo chorioallantoic membrane

Photodynamic therapy (PDT) induces damage to the endothelium, which can lead to increased vascular permeability and, under intensive PDT conditions, even to platelet aggregation, vasoconstriction, and blood flow stasis. Eventually, ischemia, hypoxia, and inflammation can occur, resulting in angiogenesis. We studied the sequence of the vascular events after Visudyne®-PDT in the chicken chorioallantoic membrane (CAM) at day 11 of development. Using epi-fluorescence microscopy, we monitored the regrowth of capillaries in the PDT treated area. Immediately after irradiation, the treatment resulted in blood flow arrest. And 24 h post PDT, sprouting of new blood vessels was observed at the edge of the PDT zone. Neovessels looping out from the edge of the PDT zone gave rise to specialized endothelial tip structures guiding the vessels towards the center of the treated area. At 48 h almost all of the treated area was repopulated with functional but morphologically altered vasculature. These observations also showed reperfusion of some of the vessels that had been closed by the PDT treatment. CAM samples were immunohistochemically stained for Ki-67 showing proliferation of endothelial cells in the PDT area. Also, several markers of immature and angiogenic blood vessels, such as αVβ3-integrin, vimentin and galectin-1, were found to be enhanced in the PDT area, while the endothelial maturation marker intercellular adhesion molecule (ICAM)-1 was found to be suppressed. These results demonstrate that the new vascular bed is formed by both neo-angiogenesis and reperfusion of existing vessels. Both the quantitative real-time RT–PCR profile and the response to pharmacological treatment with Avastin®, an inhibitor of angiogenesis, suggest that angiogenesis occurs after PDT. The observed molecular profiling results and the kinetics of gene regulation may enable optimizing combination therapies involving PDT for treatment of cancer and other diseases

Suppression of Sproutys Has a Therapeutic Effect for a Mouse Model of Ischemia by Enhancing Angiogenesis

Sprouty proteins (Sproutys) inhibit receptor tyrosine kinase signaling and control various aspects of branching morphogenesis. In this study, we examined the physiological function of Sproutys in angiogenesis, using gene targeting and short-hairpin RNA (shRNA) knockdown strategies. Sprouty2 and Sprouty4 double knockout (KO) (DKO) mice were embryonic-lethal around E12.5 due to cardiovascular defects. The number of peripheral blood vessels, but not that of lymphatic vessels, was increased in Sprouty4 KO mice compared with wild-type (WT) mice. Sprouty4 KO mice were more resistant to hind limb ischemia and soft tissue ischemia than WT mice were, because Sprouty4 deficiency causes accelerated neovascularization. Moreover, suppression of Sprouty2 and Sprouty4 expression in vivo by shRNA targeting accelerated angiogenesis and has a therapeutic effect in a mouse model of hind limb ischemia. These data suggest that Sproutys are physiologically important negative regulators of angiogenesis in vivo and novel therapeutic targets for treating peripheral ischemic diseases

Retinoic Acid Mediates Regulation of Network Formation by COUP-TFII and VE-Cadherin Expression by TGFβ Receptor Kinase in Breast Cancer Cells

Tumor development, growth, and metastasis depend on the provision of an adequate vascular supply. This can be due to regulated angiogenesis, recruitment of circulating endothelial progenitors, and/or vascular transdifferentiation. Our previous studies showed that retinoic acid (RA) treatment converts a subset of breast cancer cells into cells with significant endothelial genotypic and phenotypic elements including marked induction of VE-cadherin, which was responsible for some but not all morphological changes. The present study demonstrates that of the endothelial-related genes induced by RA treatment, only a few were affected by knockdown of VE-cadherin, ruling it out as a regulator of the RA-induced endothelial genotypic switch. In contrast, knockdown of the RA-induced gene COUP-TFII prevented the formation of networks in Matrigel but had no effect on VE-cadherin induction or cell fusion. Two pan-kinase inhibitors markedly blocked RA-induced VE-cadherin expression and cell fusion. However, RA treatment resulted in a marked and broad reduction in tyrosine kinase activity. Several genes in the TGFβ signaling pathway were induced by RA, and specific inhibition of the TGFβ type I receptor blocked both RA-induced VE-cadherin expression and cell fusion. Together these data indicate a role for the TGFβ pathway and COUP-TFII in mediating the endothelial transdifferentiating properties of RA

Deciphering von Hippel-Lindau (VHL/Vhl)-Associated Pancreatic Manifestations by Inactivating Vhl in Specific Pancreatic Cell Populations

The von Hippel-Lindau (VHL) syndrome is a pleomorphic familial disease characterized by the development of highly vascularized tumors, such as hemangioblastomas of the central nervous system, pheochromocytomas, renal cell carcinomas, cysts and neuroendocrine tumors of the pancreas. Up to 75% of VHL patients are affected by VHL-associated pancreatic lesions; however, very few reports in the published literature have described the cellular origins and biological roles of VHL in the pancreas. Since homozygous loss of Vhl in mice resulted in embryonic lethality, this study aimed to characterize the functional significance of VHL in the pancreas by conditionally inactivating Vhl utilizing the Cre/LoxP system. Specifically, Vhl was inactivated in different pancreatic cell populations distinguished by their roles during embryonic organ development and their endocrine lineage commitment. With Cre recombinase expression directed by a glucagon promoter in α-cells or an insulin promoter in β-cells, we showed that deletion of Vhl is dispensable for normal functions of the endocrine pancreas. In addition, deficiency of VHL protein (pVHL) in terminally differentiated α-cells or β-cells is insufficient to induce pancreatic neuroendocrine tumorigenesis. Most significantly, we presented the first mouse model of VHL-associated pancreatic disease in mice lacking pVHL utilizing Pdx1-Cre transgenic mice to inactivate Vhl in pancreatic progenitor cells. The highly vascularized microcystic adenomas and hyperplastic islets that developed in Pdx1-Cre;Vhl f/f homozygous mice exhibited clinical features similar to VHL patients. Establishment of three different, cell-specific Vhl knockouts in the pancreas have allowed us to provide evidence suggesting that VHL is functionally important for postnatal ductal and exocrine pancreas, and that VHL-associated pancreatic lesions are likely to originate from progenitor cells, not mature endocrine cells. The novel model systems reported here will provide the basis for further functional and genetic studies to define molecular mechanisms involved in VHL-associated pancreatic diseases