Continuous hydroxylation of the HIF-1 transcription factor ␣ subunit by oxygen and 2-oxoglutarate-dependent dioxygenases promotes decay of this protein and thus prevents the transcriptional activation of many genes involved in energy metabolism, angiogenesis, cell survival, and matrix modification. Hypoxia blocks HIF-1␣ hydroxylation and thus activates HIF-1␣-mediated gene expression. Several nonhypoxic stimuli can also activate HIF-1, although the mechanisms involved are not well known. Here we show that the glucose metabolites pyruvate and oxaloacetate inactivate HIF-1␣ decay in a manner selectively reversible by ascorbate, cysteine, histidine, and ferrous iron but not by 2-oxoglutarate or oxygen. Pyruvate and oxaloacetate bind to the 2-oxoglutarate site of HIF-1␣ prolyl hydroxylases, but their effects on HIF-1 are not mimicked by other Krebs cycle intermediates, including succinate and fumarate. We show that inactivation of HIF-1 hydroxylation by glucose-derived 2-oxoacids underlies the prominent basal HIF-1 activity commonly seen in many highly glycolytic cancer cells. Since HIF-1 itself promotes glycolytic metabolism, enhancement of HIF-1 by glucose metabolites may constitute a novel feed-forward signaling mechanism involved in malignant progression.Mammalian cells adapt to hypoxia through the action of the heterodimeric transcription factor HIF-1. Such adaptations can also promote carcinogenesis by inducing angiogenesis, treatment resistance, and invasiveness in hypoxic cancer cells within tumors (1). In the presence of oxygen, the HIF-1␣ subunit undergoes rapid decay via a ubiquitin-proteasome degradation pathway involving the von HippelLindau tumor suppressor gene product pVHL (2-4). The binding of pVHL to HIF-1␣ requires the post-translational hydroxylation of proline residues (Pro 402 and Pro 564 ) within the HIF-1␣ oxygen-dependent degradation (ODD) 4 domain (5, 6). This modification is prevented during hypoxia, thus allowing HIF-1␣ to escape proteolysis, dimerize with HIF-1␤, and translocate to the nucleus. A separately controlled, O 2 -dependent hydroxylation of asparagine 803 in the HIF-1␣ C-terminal transactivation domain inhibits HIF-1 interaction with the p300/CBP coactivator, thereby blocking HIF-1 transcriptional activity in the presence of oxygen (7,8). Three HIF-1␣ prolyl hydroxylases (HPH1 to -3; also referred to as PHD3 to -1, respectively) and one O 2 -dependent HIF-1␣ asparaginyl hydroxylase (factor inhibiting HIF, or FIH) have been clearly identified so far (9 -11). These enzymes are all members of the 2-oxoglutarate-dependent family of dioxygenases and have an absolute requirement for oxygen, ferrous iron, and 2-oxoglutarate (2-OG). This explains how hypoxia, iron chelators such as desferrioxamine (DFO), and artificial 2-OG analogs such as N-oxalylglycine or its cellpermeable precursor dimethyloxalylglycine (DMOG) can all prevent HIF-1␣ proteolysis and activate HIF-mediated gene expression. Ascorbate is also required for the sustained activity of many 2-OG-dependent dioxygenases (12, 1...

Hypoxia-inducible autocrine and paracrine EPO signaling participates in the malignant progression of GBMs.

Airway epithelial cells are a key barrier to inhaled toxicants, pollutants, and infectious agents. During severe epithelial injury and in transplantation settings, it would be desirable to rapidly reintroduce a functional epithelium through the engraftment of exogenous cells or by promoting host regeneration. However, the threedimensional surface of the airway and the requirement to maintain airflow using stents, which cause mechanical shear at the tracheal surface, are challenges. In this study, we investigate a graft composed of human airway epithelial stem/progenitor cells and lung fibroblasts embedded within a supportive collagen matrix, which we propose might be more resistant to mechanical shear than cells exposed directly to the lumen surface. Cells were combined in a collagen I hydrogel before it was dehydrated into a mechanically stable sheet using RAFT TM absorbers. After 48 h, KRT5+ spheroids formed and bromodeoxyuridine staining indicated active proliferation. To test whether epithelial cell-and fibroblast-containing grafts were able to successfully engraft on a section of trachea in vivo, they were implanted onto revascularized, decellularized tracheal scaffolds in a rabbit model. After 1 week, constructs had engrafted with signs of revascularization and keratin-positive cells were found throughout the scaffold. Although the long-term fate of these cells in vivo remains uncertain, we envisage that this strategy could improve host epithelial repair and/or contribute directly to mucosal regeneration.

Engineering tissue structures that mimic those found in vivo remains a challenge for modern biology. We demonstrate a new technique for engineering composite structures of cells comprising layers of heterogeneous cell types. An acoustofluidic bioreactor is used to assemble epithelial cells into a sheet-like structure. On transferring these cell sheets to a confluent layer of fibroblasts, the epithelial cells cover the fibroblast surface by collective migration maintaining distinct epithelial and fibroblast cell layers. The collective behaviour of the epithelium is dependent on the formation of cell-cell junctions during levitation and contrasts with the behaviour of mono-dispersed epithelial cells where cell-matrix interactions dominate and hinder formation of discrete cell layers. The multilayered tissue model is shown to form a polarised epithelial barrier and respond to apical challenge. The method is useful for engineering a wide range of layered tissue types and mechanistic studies on collective cell migration.

Development of synthetic surfaces that are highly reproducible and biocompatible for in vitro cell culture offers potential for development of improved models for studies of cellular physiology and pathology. They may also be useful in tissue engineering by removal of the need for biologically-derived components such as extracellular matrix proteins. We synthesised four types of 2-alkyl-2-oxazoline polymers ranging from the hydrophilic poly(2-methyl-2-oxazoline) to the hydrophobic poly(2-n-butyl-2-oxazoline). The polymers were terminated using amine-functionalised glass coverslips, enabling the synthetic procedure to be reproducible and scaleable. The polymer-coated glass slides were tested for biocompatibility using human epithelial (16HBE 14o-) and fibroblastic (MRC5) cell lines.Differences in adhesion and motility of the two cell types was observed, with the poly(2-isopropyl-2-oxazoline) polymer equally supporting the growth of both cell types, whereas poly(2-n-butyl-2-oxazoline) showed selectivity for fibroblast growth. In summary, 2-alkyl-2-oxazoline polymers may be a useful tool for building in vitro model cell culture models with preferential adhesion of specific cell types.