Heparanase overexpression reduces hepcidin expression, affects iron homeostasis and alters the response to inflammation

Hepcidin is the key regulator of systemic iron availability that acts by controlling the degradation of the iron exporter ferroportin. It is expressed mainly in the liver and regulated by iron, inflammation, erythropoiesis and hypoxia. The various agents that control its expression act mainly via the BMP6/SMAD signaling pathway. Among them are exogenous heparins, which are strong hepcidin repressors with a mechanism of action not fully understood but that may involve the competition with the structurally similar endogenous Heparan Sulfates (HS). To verify this hypothesis, we analyzed how the overexpression of heparanase, the HS degrading enzyme, modified hepcidin expression and iron homeostasis in hepatic cell lines and in transgenic mice. The results showed that transient and stable overexpression of heparanase in HepG2 cells caused a reduction of hepcidin expression and of SMAD5 phosphorylation. Interestingly, the clones showed also altered level of TfR1 and ferritin, indices of a modified iron homeostasis. The heparanase transgenic mice showed a low level of liver hepcidin, an increase of serum and liver iron with a decrease in spleen iron content. The hepcidin expression remained surprisingly low even after treatment with the inflammatory LPS. The finding that modification of HS structure mediated by heparanase overexpression affects hepcidin expression and iron homeostasis supports the hypothesis that HS participate in the mechanisms controlling hepcidin expression

Atomic-resolution spectroscopic imaging of ensembles of nanocatalyst particles across the life of a fuel cell

The thousandfold increase in data-collection speed enabled by aberration-corrected optics allows us to overcome an electron microscopy paradox - how to obtain atomic-resolution chemical structure in individual nanoparticles, yet record a statistically significant sample from an inhomogeneous population. This allowed us to map hundreds of Pt-Co nanoparticles to show atomic-scale elemental distributions across different stages of the catalyst aging in a proton-exchange-membrane fuel cell, and relate Pt-shell thickness to treatment, particle size, surface orientation, and ordering.Comment: 28 pages, 5 figures, accepted, nano letter

HemaMax™, a Recombinant Human Interleukin-12, Is a Potent Mitigator of Acute Radiation Injury in Mice and Non-Human Primates

HemaMax, a recombinant human interleukin-12 (IL-12), is under development to address an unmet medical need for effective treatments against acute radiation syndrome due to radiological terrorism or accident when administered at least 24 hours after radiation exposure. This study investigated pharmacokinetics, pharmacodynamics, and efficacy of m-HemaMax (recombinant murine IL-12), and HemaMax to increase survival after total body irradiation (TBI) in mice and rhesus monkeys, respectively, with no supportive care. In mice, m-HemaMax at an optimal 20 ng/mouse dose significantly increased percent survival and survival time when administered 24 hours after TBI between 8–9 Gy (p<0.05 Pearson's chi-square test). This survival benefit was accompanied by increases in plasma interferon-γ (IFN-γ) and erythropoietin levels, recovery of femoral bone hematopoiesis characterized with the presence of IL-12 receptor β2 subunit–expressing myeloid progenitors, megakaryocytes, and osteoblasts. Mitigation of jejunal radiation damage was also examined. At allometrically equivalent doses, HemaMax showed similar pharmacokinetics in rhesus monkeys compared to m-HemaMax in mice, but more robustly increased plasma IFN-γ levels. HemaMax also increased plasma erythropoietin, IL-15, IL-18, and neopterin levels. At non-human primate doses pharmacologically equivalent to murine doses, HemaMax (100 ng/Kg and 250 ng/Kg) administered at 24 hours after TBI (6.7 Gy/LD50/30) significantly increased percent survival of HemaMax groups compared to vehicle (p<0.05 Pearson's chi-square test). This survival benefit was accompanied by a significantly higher leukocyte (neutrophils and lymphocytes), thrombocyte, and reticulocyte counts during nadir (days 12–14) and significantly less weight loss at day 12 compared to vehicle. These findings indicate successful interspecies dose conversion and provide proof of concept that HemaMax increases survival in irradiated rhesus monkeys by promoting hematopoiesis and recovery of immune functions and possibly gastrointestinal functions, likely through a network of interactions involving dendritic cells, osteoblasts, and soluble factors such as IL-12, IFN-γ, and cytoprotectant erythropoietin

Transgenic mice overexpressing heparanase have increased ferritin-iron and ferritin protein content in the liver.

<p>(A and B) Western blot of liver extracts from WT and TG-HPA mice (A) for ferritin L-chain (FTL) subunits in SDS-PAGE with GAPDH as calibrator and (B) for assembled ferritin in non-denaturing PAGE. (C) Prussian blue stain of non-denaturing PAGE loaded with 50 ug protein, before (upper) and after enhancing with DAB and H₂O₂ (lower). rFTL is control purified recombinant mouse FTL. (D) Western blot of Ferroportin (FPN) and (E) of Transferrin Receptor1 (TfR1) and their respective GAPDH as calibrator. Densitometry data were obtained from 3 independent experiments.</p

Treatment with heparin and IL6 of HepG2 clones overexpressing heparanase.

<p>(A) The two HepG2 clones overexpressing HPA (HPA3 and HPA6) and control (MOCK) cells were treated with 0.12 μg/mL of RO-82 heparin for 6 h and hepcidin mRNA evaluated. (B) The clones and control cells were treated with 50 ng/mL IL6 for 3 h and analyzed for mRNA level of Socs3 and (C) hepcidin in relation to Hprt1. The values are expressed as fold change of their respective untreated controls (-). (D) Western blot of pSMAD5, total SMAD5, pSTAT3, total STAT3 and of ACTIN as housekeeping.</p

Transgenic mice overexpressing Heparanase showed increased BMP6/SMAD signaling.

<p>(A) evaluation of BMP6 mRNA in the liver of WT and TG-HPA mice (B) evaluation of Id1 mRNA, in relation to Hprt1. (C) Western blot of pSMAD5, of total SMAD5 and of GAPDH as housekeeping for normalization in densitometry quantification. The values are expressed as fold change of wild type mice.</p

Transgenic mice overexpressing heparanase showed reduced liver hepcidin mRNA and serum protein.

<p>(A) Liver hepcidin mRNA levels of wild type (WT) and transgenic HPA mice (TG-HPA) normalized to Hprt1. The values are expressed as fold change of wild type mice. (B) Quantification of serum hepcidin by SELDI-TOF in the same mice.</p

HepG2 clones overexpressing heparanase showed a reduction of hepcidin expression and indices of iron loading.

<p>Two stable clones of HepG2 cells transfected with pcDNA3.1-HPA (HPA3 and HPA6) were analyzed for hepcidin expression, BMP/SMAD signaling and indices of iron status. (A) qPCR was assessed to analyze the level of HPA mRNA. (B) Western blot for HPA shows the level of the latent (65 kDa) and active (50 kDa) forms. Densitometry quantification of the two forms was performed in relation to actin as calibrator. (C) qPCR was performed to analyze the level of hepcidin mRNA, (D) and the level of Id1 mRNA in relation to Hprt1. (E) WB of phosphorylated SMAD5 and their densitometry quantification referred to actin and WB of total SMAD5, (F) WB of transferrin Receptor 1, Tfr1, and its densitometry, (G) WB of ferritin light chain, FTL and its densitometry; (H) WB of Ferroportin (FPN) and its densitometry. The values are expressed as–dCt (for HPA mRNA) or as fold change over the control (MOCK) (for hepcidin and Id1 mRNA). The images are representative from three different analyses</p

HepG2 cells transiently transfected with heparanase showed a reduction of hepcidin mRNA.

<p>HepG2 cells were transfected with pcDNA3.1-HPA plasmid (HPA) or empty pcDNA3.1 as control (MOCK) and harvested 48 h after the transfection. (A) Relative level of HPA mRNA was measured by qRT-PCR (B) Western blot of SDS-PAGE with anti-HPA antibodies show the levels of its latent (65 kDa) and active (50 kDa) form. Densitometry quantification of the two protein forms was performed in relation to Actin. (C) The level of hepcidin mRNA and (D) Id1 mRNA was analyzed by qPCR and normalized for Hprt1. (E) The phosphorylated (pSMAD5) and total SMAD5 were analyzed by western blot and pSMAD5 densitometry was normalized to actin. In (A) the values are expressed as–dCt for HPA mRNA, in C and D as fold change over the control (MOCK) for hepcidin and Id1 mRNA., respectively</p

Transgenic mice overexpressing heparanase showed altered iron homeostasis.

<p>(A) Levels of serum iron in the WT and TG-HPA mice, (B) non-heme liver iron and (C) non-heme spleen iron levels, measured by a spectrophotometric assay. (D) Perl’s stain of spleen sections that showed a lower number of iron granules in TG-HPA compared to WT mice. The sections were counterstained with hematoxylin and eosin</p