Angiopoietins are ligands for the endothelial cell tyrosine kinase receptor Tie-2. Ang-1, the major physiological activator of Tie-2, promotes blood vessel maturation and stability. Ang-2 counteracts this effect by competitively inhibiting the binding of Ang-1 to Tie-2. Using a combined RNase protection/semiquantitative reverse transcriptase-polymerase chain reaction approach, we demonstrate that hypoxia up-regulates Ang-2 mRNA levels by up to 3.3-fold in two human endothelial cell lines. In bovine microvascular endothelial (BME) cells, the flavoprotein oxidoreductase inhibitor diphenylene iodonium (DPI) and the related compound iodonium diphenyl mimic induction of Ang-2 but not vascular endothelial growth factor (VEGF) by hypoxia; in combination with hypoxia, DPI further increases Ang-2 expression but has no effect on the induction of VEGF by hypoxia. Neither Ang-2 or VEGF was increased by cyanide or rotenone, suggesting that failure in mitochondrial electron transport is not involved in the oxygen-sensing system that controls their expression. In ischemic rat dorsal skin flaps or in the brain of rats maintained for 12 hours under conditions of hypoxia, Ang-2 mRNA was up-regulated 7.5-or 17.6-fold, respectively. VEGF was concomitantly increased, whereas expression of Ang-1, Tie-2, and the related receptor Tie-1 was unaltered. In situ hybridization localized Ang-2 mRNA to endothelial cells in hypoxic skin. These findings 1) show that up-regulation of Ang-2 by hypoxia occurs widely in endothelial cells in vitro and in vivo; 2) suggest that induction of Ang-2, but not VEGF, by hypoxia in BME cells is controlled by a flavoprotein oxidoreductase that is sensitive to iodonium compounds Angiogenesis, the sprouting of new capillary blood vessels from preexisting microvasculature, is an absolute requirement for the development and maintenance of tissues and organs and is an important component in a number of pathological settings, including diabetic retinopathy, rheumatoid arthritis, and tumor growth. During angiogenesis, endothelial cells loose their intimate contacts with adjacent pericytes, degrade their underlying basement membrane, and migrate into the surrounding stroma as cell cords (sprouting phase). Later these cords form a lumen, deposit a new basement membrane, develop cell-cell junctions, and recruit perivascular cells (maturation phase), thus resulting in new functional vessels. 1,2As might have been predicted, the search for angiogenic regulators has resulted in the discovery of a number of molecules, including polypeptide growth factors (vascular endothelial growth factors, angiopoietins, fibroblast growth factors, transforming growth factors-␣ and -␤, platelet-derived growth factor-BB, tumor necrosis factor-␣, hepatocyte growth factor), chemokines (platelet factor 4), enzymes (angiogenin, platelet-derived endothelial cell growth factor/thymidine phosphorylase), extracellular matrix components/coagulation factors or fragments thereof (thrombospondin, endostatin, 29 kdfibronectin fragment, angiostatin), ad...

(R)‐1,1′‐Binaphthyl‐2,2′‐diol (R‐BINOL) derived chiral phosphoric acids have been explored as organocatalysts for the asymmetric oxidation of a series of aryl alkyl sulfides and 1,3‐dithianes derived from aldehydes with aqueous hydrogen peroxide (H2O2) as the terminal oxidant. The enantiomerically enriched sulfoxides are obtained in moderate to excellent yield (up to 99%) with excellent diastereoselectivity (up to >99:1 dr) and moderate to good enantioselectivity (up to 91:9 er). In particular, the present protocol stereoselectively provides an efficient access to enantiomerically enriched aryl alkyl sulfoxides and dithioacetal mono‐sulfoxides, which strictly restrains the formation of the undesirable by‐products: sulfones or disulfoxides. The tracking experiments also verify that this approach proceeds via a direct sulfoxidation process, instead of a kinetic resolution route by overoxidation of the resulting sulfoxides.

Mutations in the human fibrinogen genes can lead to the absence of circulating fibrinogen and cause congenital afibrinogenemia. This rare bleeding disorder is associated with a variable phenotype, which may be influenced by environment and genotype. Here, we present a zebrafish model of afibrinogenemia. We introduced targeted mutations into the zebrafish fga gene using zinc finger nuclease technology. Animals carrying 3 distinct frameshift mutations in fga were raised and bred to produce homozygous mutants. Using a panel of anti-zebrafish fibrinogen antibodies, fibrinogen was undetectable in plasma preparations from homozygous mutant fish. We observed hemorrhaging in fga mutants and reduced survival compared with control animals. This model will now serve in the search for afibrinogenemia modifying genes or agents and, to our knowledge, is the first transmissible zebrafish model of a defined human bleeding disorder

Congenital afibrinogenemia is a rare bleeding disorder characterized by the absence in circulation of fibrinogen, a hexamer composed of two sets of three polypeptides (Aalpha, Bbeta and gamma). Each polypeptide is encoded by a distinct gene, FGA, FGB and FGG, all three clustered in a region of 50 kb on 4q31. A subset of afibrinogenemia mutations has been shown to specifically impair fibrinogen secretion, but the underlying molecular mechanisms remained to be elucidated. Here, we show that truncation of the seven most C-terminal residues (R455-Q461) of the Bbeta chain specifically inhibits fibrinogen secretion. Expression of additional mutants and structural modelling suggests that neither the last six residues nor R455 is crucial per se for secretion, but prevent protein misfolding by protecting hydrophobic residues in the betaC core. Immunofluorescence and immuno-electron microscopy studies indicate that secretion-impaired mutants are retained in a pre-Golgi compartment. In addition, expression of Bbeta, gamma and angiopoietin-2 chimeric molecules demonstrated that the betaC domain prevents the secretion of single chains and complexes, whereas the gammaC domain allows their secretion. Our data provide new insight into the mechanisms accounting for the quality control of fibrinogen secretion and confirm that mutant fibrinogen retention is one of the pathological mechanisms responsible for congenital afibrinogenemia.

Plasma fibrinogen molecules comprise 2 copies of Aα, Bβ, and γ chains folded into a hexameric protein. A minor fibrinogen isoform with an extended Aα chain (AαE) is more abundant in newborn human blood than in adults. Larval zebrafish produce predominantly AαE-containing fibrinogen, but its functional significance is unclear. In 3-day-old zebrafish, when hemostasis is reliant on fibrinogen and erythrocyte-rich clotting but is largely thrombocyte-independent, we measured the time to occlusion (TTO) in a laser-induced venous thrombosis assay in 3 zebrafish strains (AB, TU, and AB × TL hybrids). AB larvae showed delayed TTO compared with the TU and AB × TL strains. Mating AB with TU or TL produced larvae with a TU-like TTO. In contrast to TU, AB larvae failed to produce fibrinogen AαE, due to a mutation in the AαE-specific coding region of fibrinogen α-chain gene (fga). We investigated whether the lack of AαE explained the delayed AB TTO. Transgenic expression of AαE, but not Aα, shortened the AB TTO to that of TU. AαE rescued venous occlusion in fibrinogen mutants or larvae with morpholino-targeted fibrinogen α-chain messenger RNA, but Aα was less effective. In 5-day-old larvae, circulating thrombocytes contribute to hemostasis, as visualized in Tg(itga2b:EGFP) transgenics. Laser-induced venous thrombocyte adhesion and aggregation is reduced in fibrinogen mutants, but transgenic expression of Aα or AαE restored similar thrombocyte accumulation at the injury site. Our data demonstrate a genetic modifier of venous thrombosis and a role for fibrinogen AαE in early developmental blood coagulation, and suggest a link between differentially expressed fibrinogen isoforms and the cell types available for clotting.