A long-term "memory" of HIF induction in response to chronic mild decreased oxygen after oxygen normalization

Background Endothelial dysfunction (ED) is functionally characterized by decreased vasorelaxation, increased thrombosis, increased inflammation, and altered angiogenic potential, has been intimately associated with the progression and severity of cardiovascular disease. Patients with compromised cardiac function oftentimes have a state of chronic mild decreased oxygen at the level of the vasculature and organs, which has been shown to exacerbate ED. Hypoxia inducible factor (HIF) is a transcription factor complex shown to be the master regulator of the cellular response to decreased oxygen levels and many HIF target genes have been shown to be associated with ED. Methods Human endothelial and aortic smooth muscle cells were exposed either to A) normoxia (21% O2) for three weeks, or to B) mild decreased oxygen (15% O2) for three weeks to mimic blood oxygen levels in patients with heart failure, or to C) mild decreased oxygen for two weeks followed by one week of normoxia ("memory" treatment). Levels of HIF signaling genes (HIF-1α, HIF-2α, VEGF, BNIP3, GLUT-1, PAI-1 and iNOS) were measured both at the protein and mRNA levels. Results It was found that chronic exposure to mild decreased oxygen resulted in significantly increased HIF signaling. There was also a "memory" of HIF-1α and HIF target gene induction when oxygen levels were normalized for one week, and this "memory" could be interrupted by adding a small molecule HIF inhibitor to the last week of normalized oxygen. Finally, levels of ubiquitylated HIF-1α were reduced in response to chronic mild decreased oxygen and were not full restored after oxygen normalization. Conclusion These data suggest that HIF signaling may be contributing to the pathogenesis of endothelial dysfunction and that normalization of oxygen levels may not be enough to reduce vascular stress

Synthesis of Uridine 5′-diphosphoiduronic Acid: A Potential Substrate for the Chemoenzymatic Synthesis of Heparin

An improved understanding of the biological activities of heparin requires structurally defined heparin oligosaccharides. The chemoenzymatic synthesis of heparin oligosaccharides relies on glycosyltransferases that use UDP-sugar nucleotides as donors. Uridine 5′-diphosphoiduronic acid (UDP-IdoA) and uridine 5′-diphosphohexenuronic acid (UDP-HexUA) have been synthesized as potential analogues of uridine 5′-diphosphoglucuronic acid (UDP-GlcA) for enzymatic incorporation into heparin oligosaccharides. Non-natural UDP-IdoA and UDP-HexUA were tested as substrates for various glucuronosyltransferases to better understand enzyme specificity

Heparan Sulfate Domains Required for Fibroblast Growth Factor 1 and 2 Signaling through Fibroblast Growth Factor Receptor 1c

A small library of well defined heparan sulfate (HS) polysaccharides was chemoenzymatically synthesized and used for a detailed structure-activity study of fibroblast growth factor (FGF) 1 and FGF2 signaling through FGF receptor (FGFR) 1c. The HS polysaccharide tested contained both undersulfated (NA) domains and highly sulfated (NS) domains as well as very well defined non-reducing termini. This study examines differences in the HS selectivity of the positive canyons of the FGF12-FGFR1c2 and FGF22-FGFR1c2 HS binding sites of the symmetric FGF2-FGFR2-HS2 signal transduction complex. The results suggest that FGF12-FGFR1c2 binding site prefers a longer NS domain at the non-reducing terminus than FGF22-FGFR1c2. In addition, FGF22-FGFR1c2 can tolerate an HS chain having an N-acetylglucosamine residue at its non-reducing end. These results clearly demonstrate the different specificity of FGF12-FGFR1c2 and FGF22-FGFR1c2 for well defined HS structures and suggest that it is now possible to chemoenzymatically synthesize precise HS polysaccharides that can selectively mediate growth factor signaling. These HS polysaccharides might be useful in both understanding and controlling the growth, proliferation, and differentiation of cells in stem cell therapies, wound healing, and the treatment of cancer

Chemoenzymatic Synthesis of Uridine Diphosphate-GlcNAc and Uridine Diphosphate-GalNAc Analogs for the Preparation of Unnatural Glycosaminoglycans

Eight N-acetylglucosamine-1-phosphate and N-acetylgalactosamine-1-phosphate analogs have been synthesized chemically and were tested for their recognition by the GlmU uridyltransferase enzyme. Among these, only substrates that have an amide linkage to the C-2 nitrogen were transferred by GlmU to afford their corresponding uridine diphosphate(UDP)-sugar nucleotides. Resin-immobilized GlmU showed comparable activity to non-immobilized GlmU and provides a more facile final step in the synthesis of an unnatural UDP-donor. The synthesized unnatural UDP-donors were tested for their activity as substrates for glycosyltransferases in the preparation of unnatural glycosaminoglycans in vitro. A subset of these analogs was useful as donors, increasing the synthetic repertoire for these medically important polysaccharides

Fibroblast Growth Factor-based Signaling through Synthetic Heparan Sulfate Blocks Copolymers Studied Using High Cell Density Three-dimensional Cell Printing

Four well-defined heparan sulfate (HS) block copolymers containing S-domains (high sulfo group content) placed adjacent to N-domains (low sulfo group content) were chemoenzymatically synthesized and characterized. The domain lengths in these HS block co-polymers were ∼40 saccharide units. Microtiter 96-well and three-dimensional cell-based microarray assays utilizing murine immortalized bone marrow (BaF3) cells were developed to evaluate the activity of these HS block co-polymers. Each recombinant BaF3 cell line expresses only a single type of fibroblast growth factor receptor (FGFR) but produces neither HS nor fibroblast growth factors (FGFs). In the presence of different FGFs, BaF3 cell proliferation showed clear differences for the four HS block co-polymers examined. These data were used to examine the two proposed signaling models, the symmetric FGF2-HS2-FGFR2 ternary complex model and the asymmetric FGF2-HS1-FGFR2 ternary complex model. In the symmetric FGF2-HS2-FGFR2 model, two acidic HS chains bind in a basic canyon located on the top face of the FGF2-FGFR2 protein complex. In this model the S-domains at the non-reducing ends of the two HS proteoglycan chains are proposed to interact with the FGF2-FGFR2 protein complex. In contrast, in the asymmetric FGF2-HS1-FGFR2 model, a single HS chain interacts with the FGF2-FGFR2 protein complex through a single S-domain that can be located at any position within an HS chain. Our data comparing a series of synthetically prepared HS block copolymers support a preference for the symmetric FGF2-HS2-FGFR2 ternary complex model

Mutant p53 facilitates pro-angiogenic, hyperproliferative phenotype in response to chronic relative hypoxia

There is much controversy in the literature regarding the role of p53 status response on hypoxia inducible factor (HIF) signaling in response to chronic relative hypoxia (CRH). The goal of this paper was to methodically examine this response in isogenically matched tumor cells. We report that p53-mutant (MUT) cells, versus p53-wild-type (WT) cells, showed decreased apoptosis, increased cell proliferation with higher basal HIF-1 alpha levels in response to CRH. In addition, we found increased HIF-mediated transactivation and increased VEGF release with decreased HIF-1 alpha/p53 and HIF-1 alpha/ MDM-2 partnering in p53-MUT versus p53-WT cells in response to CRH. (c) 2006 Elsevier Ireland Ltd. All rights reserved