ALBERT

Description: Erythropoietin (Epo) gene expression is under the control of hypoxia-inducible factor 1 (HIF-1), and is negatively regulated by GATA. Interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α), which increase the binding activity of GATA and inhibit Epo promoter activity, are increased in patients with anemia of chronic disease (ACD). We previously demonstrated the ability of K-7174 (a GATA-specific inhibitor), when injected intraperitoneally, to improve Epo production that had been inhibited by IL-1β or TNF-α treatment. In the present study, we examined the ability of both K-11706, which inhibits GATA and enhances HIF-1 binding activity, and K-13144, which has no effect on GATA or HIF-1 binding activity, to improve Epo production following inhibition by IL-1β or TNF-α in Hep3B cells in vitro and in an in vivo mouse assay. Oral administration of K-11706 reversed the decreases in hemoglobin and serum Epo concentrations, reticulocyte counts, and numbers of erythroid colony-forming units (CFU-Es) induced by IL-1β or TNF-α. These results raise the possibility of using orally administered K-11706 for treating patients with ACD.

Description: Erythropoietin (Epo) gene expression is controlled by hypoxia-inducible factor-1 (HIF-1); it is positively regulated through the HIF-1 binding site in the Epo enhancer and negatively regulated by GATA, which binds to the GATA site in the Epo promoter. Drugs that inhibit GATA or activate HIF-1 might increase the production of Epo and restore hemoglobin concentration. Epo gene doping and the illicit use of HIF-PHD inhibitor (FG-2216) and GATA inhibitor (K-11706) or HIF-1 activators may be used as new doping practices to increase the number of red blood cells. The broad scope of this research is to develop a system for detecting illegal hypoxia-inducible gene manipulation. We performed hematologic analyses, a treadmill exercise test, microarray and real-time RT-PCR (reverse transcriptase-polymerase chain reaction) to examine the effects of K-11706 and FG-2216 on mice, and compared these effects with those induced by recombinant human Epo (rhEpo) as a positive control. Oral administration of K-11706 for 5 and 8 days, FG-2216 for 5 days, and 14-day intra-peritoneal injection of rhEpo on alternate days significantly increased Epo and hemoglobin concentrations, hematocrit, and endurance performance, respectively, compared with the control. Transgenic lines of mice were generated to evaluate erythropoiesis in living mice. Firefly luciferase reporter line generated with the human β-globin locus control region (Hbb-LCR) is referred as Hbb-LCR-Luc; this mouse model provides a sensitive, noninvasive, and real-time method to monitor erythropoiesis in vivo in response to the administration of K-11706 and FG-2216. The oral administration of K-11706 and FG-2216 for 5 days significantly accumulated Hbb-LCR-Luc activity in the spleen. DNA chip technology was used to investigate the molecular mechanisms responsible for hypoxia-inducible gene manipulation. Total RNA was prepared from the bone marrow cells of mice treated with K-11706 for 28 days, FG-2216 for 5 days, and rhEpo for 14 days, and in untreated mice. The genes that appeared to exhibit a difference in the expression level (increase more or decrease less than five times for K-11706 and rhEpo, two times for FG-2216) were 251 genes for K-11706, 343 genes for FG-2216, and 142 genes for rhEpo. Twenty-eight genes showed a significant difference between the 3 groups, and 4 genes showed it with K-11706 and FG-2216. Among them, real-time RT-PCR revealed that FG-2216 significantly decreased the expression levels of FOS, OSM, IL8rb, Ms4a8a, and Arl5c genes to 0.58, 0.54, 0.56, 0.46, and 0.53-fold compared with the control, respectively. A panel of several candidate genes to detect hypoxia-inducible gene manipulation by real-time RT-PCR will provide a new detection system.

Description: Key Points Mucin-type O-glycans are required for terminal differentiation of megakaryocytes and platelet production. The expression of GPIbα protein is strongly reduced in O-glycans–defective megakaryocytes and platelets.

Description: The early blood vessels of the embryo and yolk sac in mammals develop by aggregation of de novo–forming angioblasts into a primitive vascular plexus, which then undergoes a complex remodeling process. Angiogenesis is also important for disease progression in the adult. However, the precise molecular mechanism of vascular development remains unclear. It is therefore of great interest to determine which genes are specifically expressed in developing endothelial cells (ECs). Here, we used Flk1-deficient mouse embryos, which lack ECs, to perform a genome-wide survey for genes related to vascular development. We identified 184 genes that are highly enriched in developing ECs. The human orthologs of most of these genes were also expressed in HUVECs, and small interfering RNA knockdown experiments on 22 human orthologs showed that 6 of these genes play a role in tube formation by HUVECs. In addition, we created Arhgef15 knockout and RhoJ knockout mice by a gene-targeting method and found that Arhgef15 and RhoJ were important for neonatal retinal vascularization. Thus, the genes identified in our survey show high expression in ECs; further analysis of these genes should facilitate our understanding of the molecular mechanisms of vascular development in the mouse.

Description: Hypoxia-inducible factor 1 (HIF-1) plays an essential role in tumor angiogenesis and growth by regulating the transcription of several genes in response to hypoxic stress and changes in growth factors. HIF-1 is a heterodimeric transcriptional activator and consists of inducible α and constitutive β subunits. In oxygenated cells, intracellular oxygen concentrations are directly sensed by proteins containing the prolyl hydroxylase domain (PHD), which tag HIF-1α subunits for polyubiquitination and proteasomal degradation by prolyl hydroxylation using 2-oxoglutarate (2-OX) and dioxygen. Our recent studies have shown that 2-OX reduces HIF-1α, erythropoietin, and vascular endothelial growth factor (VEGF) expression by hypoxia in the hepatoma cell line Hep3B (Matsumoto K et al.; J. Cell. Physiol., 2006). Similar results were obtained in Lewis lung cancer (LLC) cells in in vitro studies. Here, to address the clinical usefulness of 2-OX, we investigated its antitumor effect using a mouse dorsal air sac (DAS) assay and a murine tumor xenograft model. For the DAS assay, LLC cells suspended in PBS with 0, 7.5, or 15 mM 2-OX per Millipore chamber were implanted subcutaneously into C57BL/6J mice. The values of blood vessel area by LLC cells were 100 ± 20.8, 64.0 ± 9.4, and 45.6 ± 4.4%, respectively, by quantitative analysis with angiogenesis-measuring software. This result indicated that 2-OX clearly inhibited the growth of subcutaneous tumors. To elucidate the effect of 2-OX on tumor growth, 2-OX was administrated to C57BL/6J mice inoculated with LLC cells. LLC cells (1 × 106) in PBS were implanted into the right flank region of 7-week-old mice. Daily intraperitoneal (i.p.) injections of 2-OX were started on the next day after implantation. From 6 to 12 days after implantation, we measured tumors with calipers and calculated volumes as (length × width2) × 0.5. LLC tumors were removed from mice 12 days after implantation for quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical studies. Tumor volumes and weights 12 days after implantation were as follows: PBS alone, 330.8 ± 108.1 mm3 and 192.6 ± 66.4 mg; 50 mg/kg 2-OX, 128.8 ± 16.4 mm3 and 111.0 ± 64.5 mg; and 100 mg/kg 2-OX, 78.7 ± 43.7 mm3 and 88.8 ± 57.5 mg. Quantitative RT-PCR revealed that 2-OX treatment (100 mg/kg) decreased the expression levels of the VEGF gene 0.67-fold in tumor tissues compared with control. We observed quantitative differences in microvessel density (PBS alone, 100 ± 16.5%; 100 mg/kg 2-OX, 46.0 ± 13.5%) using immunostaining for the endothelial cell marker CD31. Intraperitoneal injection of 2-OX significantly inhibited tumor growth and angiogenesis in tumor tissues. Combination therapy is necessary for anti-angiogenic therapy in the human. To examine the effect of 2-OX in combination with 5-fluorouracil (5-FU) chemotherapy, we injected 5-FU i.p. on day 6 and/or 2-OX i.p. on each of days 6–15 in this mouse model. Tumor volumes and weights 15 days after implantation were as follows: PBS alone, 531.2 ± 144.9 mm3 and 306.3 ± 186.6 mg; 2-OX alone, 324.0 ± 156.5 mm3 and 308.7 ± 299.0 mg; 5-FU alone, 287.1 ± 155.2 mm3 and 204.7 ± 108.9 mg; and 5-FU + 2-OX, 98.7 ± 64.9 mm3 and 130.3 ± 113.5 mg. 5-FU combined with 2-OX significantly inhibited tumor growth in this model, which was accompanied by 53% reduction of VEGF gene expression in tumor tissues removed from mice 15 days after implantation, using quantitative RT-PCR analysis. These results suggest that 2-OX is a promising anti-angiogenic therapeutic agent. To examine whether the inhibitory effect of 2-OX is specific for PHD-containing proteins, an RNAi study will be performed.

Description: The nitric oxide (NO)/cGMP pathway, by relaxing vascular smooth muscle cells, is a major physiologic regulator of tissue perfusion. We now identify thrombospondin-1 as a potent antagonist of NO for regulating F-actin assembly and myosin light chain phosphorylation in vascular smooth muscle cells. Thrombospondin-1 prevents NO-mediated relaxation of precontracted vascular smooth muscle cells in a collagen matrix. Functional magnetic resonance imaging demonstrated that an NO-mediated increase in skeletal muscle perfusion was enhanced in thrombospondin-1–null relative to wild-type mice, implicating endogenous thrombospondin-1 as a physiologic antagonist of NO-mediated vasodilation. Using a random myocutaneous flap model for ischemic injury, tissue survival was significantly enhanced in thrombospondin-1–null mice. Improved flap survival correlated with increased recovery of oxygen levels in the ischemic tissue of thrombospondin-1–null mice as measured by electron paramagnetic resonance oximetry. These findings demonstrate an important antag-onistic relation between NO/cGMP signaling and thrombospondin-1 in vascular smooth muscle cells to regulate vascular tone and tissue perfusion.

Description: In oxygenated cells, hypoxia inducible factor-1 (HIF-1)α subunits are rapidly degraded by a mechanism that involves ubiquitination by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex using 2-oxoglutarate as a substrate. This process is suppressed by hypoxia and iron chelation, allowing transcriptional activation. The interaction between human pVHL and a specific domain of the HIF-1α subunit is regulated through hydroxylation of proline residues 402 and 564 by HIF-1α proryl-hydroxylase (PHD). N-oxalyl glycine acts as a competitive inhibitor of HIF-PHDs and this inhibition is in competition with 2-oxoglutarate. We examined the effect of 2-oxoglutarate on the production of vascular endothelial growth factor (VEGF) and erythropoietin (Epo). The expression of VEGF and Epo protein were dose-dependently downregulated in Hep3B cells by the addition of 2-oxoglutarate. The enhancer activity of the HIF-1 binding site of Epo and the promoter activity of VEGF-luciferase were also dose-dependently downregulated by the addition of 2-oxoglutarate. Gel mobility shift assays revealed that the addition of 2-oxoglutarate dose-dependently inhibited HIF-1 binding activity, but did not affect GATA binding activity. Western blot analysis revealed that 2-oxoglutarate dose-dependently inhibited HIF-1α protein in Hep3B, Hela and SW480 cells in hypoxic conditions. However MG132 (the proteasome inhibitor) rescued the inhibition of HIF-1α protein expression by 2-oxoglutarate. Furthermore, under hypoxic conditions, 2-oxoglutarate dose-dependently inhibited tube formation in in vitro angiogenesis assays. These results indicate that 2-oxoglutarate treatment may be useful for the inhibition of tumor angiogenesis through decreasing HIF-1α protein, HIF-1 binding activity, the promoter activity of VEGF and enhancer activity of Epo, and the production of VEGF and Epo. More studies to determine if 2-oxoglutarate inhibits tumor angiogenesis in vivo mouse assay are in progress.