Aging is associated with increased brain iron through cortex-derived hepcidin expression

Iron is an essential molecule for biological processes, but its accumulation can lead to oxidative stress and cellular death. Due to its oxidative effects, iron accumulation is implicated in the process of aging and neurodegenerative diseases. However, the mechanism for this increase in iron with aging, and whether this increase is localized to specific cellular compartment(s), are not known. Here, we measured the levels of iron in different tissues of aged mice, and demonstrated that while cytosolic non-heme iron is increased in the liver and muscle tissue, only the aged brain cortex exhibits an increase in both the cytosolic and mitochondrial non-heme iron. This increase in brain iron is associated with elevated levels of local hepcidin mRNA and protein in the brain. We also demonstrate that the increase in hepcidin is associated with increased ubiquitination and reduced levels of the only iron exporter, ferroportin-1 (FPN1). Overall, our studies provide a potential mechanism for iron accumulation in the brain through increased local expression of hepcidin, and subsequent iron accumulation due to decreased iron export. Additionally, our data support that aging is associated with mitochondrial and cytosolic iron accumulation only in the brain and not in other tissues.

Anti-Oxidant and Anti-Inflammatory Effects of Lipopolysaccharide from Rhodobacter sphaeroides against Ethanol-Induced Liver and Kidney Toxicity in Experimental Rats

This study aimed to investigate the protective effects of lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS) against ethanol-induced hepatotoxicity and nephrotoxicity in experimental rats. The study involved an intact control group, LPS-RS group, two groups were given ethanol (3 and 5 g/kg/day) for 28 days, and two other groups (LPS-RS + 3 g/kg ethanol) and (LPS-RS + 5 g/kg ethanol) received a daily dose of LPS-RS (800 μg/kg) before ethanol. Ethanol significantly increased the expression of nuclear factor kappa B (NF-κB) and levels of malondialdehyde (MDA), tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in the liver tissue and decreased anti-oxidant enzymes. Hepcidin expression was downregulated in the liver, with increased serum levels of ferritin and iron. Prior-administration of LPS-RS alleviated the increase in oxidative stress and inflammatory markers, and preserved iron homeostasis markers. In the kidney, administration of ethanol caused significant increase in the expression of NF-κB and the levels of TNF-α and kidney injury markers; whereas LPS-RS + ethanol groups had significantly lower levels of those parameters. In conclusion; this study reports anti-oxidant, anti-inflammatory and iron homeostasis regulatory effects of the toll-like receptor 4 (TLR4) antagonist LPS-RS against ethanol induced toxicity in both the liver and the kidney of experimental rats.

The Effect of STAT3 Signal Pathway Activation on Retinopathy of Prematurity

Objective: To investigate the mechanism of activation of the signal transducer and activator of transcription 3 (STAT3) signal pathway in the process of retinopathy of prematurity (ROP).Methods: Sixty newborn Sprague-Dawley (SD) rats were randomly separated into the hyperoxia and air control groups (n = 30/in each group). The serum hepcidin level on 21 d was measured using the enzyme-linked immunosorbent assay (ELISA). The expression of HAMP and STAT3 protein in the liver was determined using reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. Retinal neovasculature was evaluated by hematoxylin and eosin (HE) stain and fluorescein lectin. The retinal endothelial cells were treated with 250 μmol/L cobalt chloride for 72 h and added S3I-201. The STAT3 level was determined by western blotting.Results: The expression of STAT3 protein increased significantly after hyperoxia stimulation. The expression of HAMP mRNA in the hyperoxia group was significantly higher than that of the control group. The proliferation of retinal cells was inhibited, and the expression of STAT3 was increased. No significant difference was noted in vascular endothelial growth factor (VEGF) mRNA. The expression of STAT3 and VEGF mRNA was significantly reduced.Conclusion: The activation of the STAT3 signal pathway increased hepcidin expression, contributing to the pathogenesis of ROP. S3I-201 inhibited the expression of STAT3 and VEGF mRNA levels. This information provides potential novel therapeutic approach to the prevention and treatment of ROP.

Treating Iron Overload Disorders with a Novel Therapeutic Antibody Targeting TMPRSS6

Iron is an essential element for almost all living organisms as it participates in a wide variety of metabolic processes. Disorders of iron metabolism are among the most prevalent human diseases, ranging from anemia to hemochromatosis. Excessive iron accumulations in major organs of iron overload patients can lead to high mortality. Hepcidin, a HAMP-encoded liver hormone, is the master regulator of iron homeostasis. By binding to the sole iron exporter ferroportin and causing internalization and degradation of the complex, hepcidin inhibits cellular iron efflux, thereby lowers plasma iron levels. Inappropriately suppressed/low hepcidin production is central to iron overload. Transmembrane protease serine-6 (TMPRSS6), a type II transmembrane serine protease primarily expressed in liver, downregulates hepcidin expression through BMP-SMAD pathway. TMPRSS6 deficiencies have been shown to cause hepcidin overexpression in both TMPRSS6-mutant mice and in patients with iron-refractory iron deficiency anemia (IRIDA). Therefore, TMPRSS6 is a viable therapeutic target for iron overload disorders. Here we report the generation of an anti-TMPRSS6 antibody through a hybridoma campaign using a DNA-based immunization approach, followed by humanization and sequence optimization. Lead antibody, hzMWTx-003 selectively binds human TMPRSS6 with low nanomolar affinity (KD: 7.6nM), and is cross-reactive to rodent (mouse and rat) and monkey (cynomolgus and rhesus) TMPRSS6. Single-dose injection of hzMWTx-003 was able to significantly elevate serum hepcidin and liver HAMP RNA levels in wildtype mice, resulting in significantly reduced serum iron level. The Hbb th3/+ mouse model of β-thalassemia, like its human counterpart, is characterized by iron overload, ineffective erythropoiesis and splenomegaly. Treatment of Hbb th3/+mice with MWTx-003 effectively increased hepcidin expression at both protein and RNA levels, leading to significantly reduced serum iron and liver non-heme iron content. MWTx-003 also dramatically improved anemia and ineffective erythropoiesis, and alleviated splenomegaly in these mice. CMC development of hzMWTx-003 confirms outstanding biophysical properties. Preliminary studies in cynomolgus monkey using GLP-grade material demonstrated good pharmacokinetics of hzMWTx-003 and expected pharmacodynamic response where reduction of serum iron could be sustained for 21 days after single dose administration. A single dose toxicology study in cynomolgus monkey revealed no safety concerns, and no production of anti-idiotype antibodies was detected. In summary, anti-TMPRSS6 antibody MWTx-003 represents a promising therapy for iron overload disorders such as β-thalassemia, and potentially other diseases where iron restriction is beneficial. Disclosures Chen: Mabwell Therapeutics Inc: Current Employment. Wang: Mabwell Therapeutics Inc: Current Employment. Zheng: Mabwell (Shanghai) Bioscience Co. Ltd: Current Employment. Huang: Mabwell Therapeutics Inc: Current Employment. Mao: Mabwell (Shanghai) Bioscience Co. Ltd.: Current Employment. Ouyang: Mabwell (Shanghai) Bioscience Co. Ltd.: Current Employment. Du: Mabwell Therapeutics Inc: Current Employment.

Obligate N-Terminal but Not C-Terminal Monoferric Transferrin Ameliorates Anemia in β-Thalassemic Mice

Transferrin (TF) is a bilobed 80kD glycoprotein with N- and C-lobe iron binding sites. TF circulates as four forms: unbound to iron (apo-TF), iron bound to the N-lobe (monoferric N-TF), the C-lobe (monoferric-C), or to both lobes (diferric-TF). Most circulating TF under physiological conditions is monoferric. The iron-bound TF forms interact with TF receptor-1 (TFR1), which is ubiquitously expressed and serves as the main mechanism for cellular iron delivery. Iron-bound TF also interacts with TF receptor-2 (TFR2) which is expressed on hepatocytes, erythroblasts, and bone cells. Whereas TFR1 serves primarily as a cargo receptor, TFR2 serves primarily to influence cellular signaling events regulating hepcidin expression, erythropoiesis, and bone formation. We proposed that different transferrin forms provide differential signaling properties in this regulation. We thus generated TF mutant mice in which all iron-containing TF was either monoferric N (Tf monoN) or monoferric C (Tf monoC). Compared with Tf monoC mice, the Tf monoN mice demonstrated increased RBC production and increased hepcidin expression relative to iron status (Parrow et al. Blood). Based on observations in β-thalassemic mice treated with exogenous TF (Li et al. Nat Med), we hypothesized that β-thalassemic mice obligate for monoN TF would demonstrate improved erythropoietic and iron parameters compared with β-thalassemic mice obligate for monoC TF. To address this hypothesis, we crossed Hbb th3/+ mice (a mouse model of β-thalassemia intermedia) with Tf monoN and Tf monoC mice. Compared with Hbb th3Tf +/+mice, in Hbb th3/+Tf monoN mice demonstrated significantly increased RBC counts, elevated hemoglobin, improved erythrocyte morphology (Figure 1A-B), decreased splenomegaly, fewer bone marrow erythroblasts, and improvement of ineffective erythropoiesis (as measured by the ratio of progenitors to RBC in the bone marrow). Additionally, serum ERFE was significantly reduced and hepcidin levels were increased in Hbb th3/+Tf monoN relative to Hbb th3/+Tf +/+controls. Conversely, hematological parameters from Hbb th3/+Tf monoC mice were comparable to Hbb th3/+Tf +/+ mice. Similarly, Hbb th3/+Tf monoCmice had no improvements in markers of ineffective erythropoiesis in the bone marrow compared with Hbb th3/+Tf +/+ mice. In summary, we demonstrate that the differential regulatory effects of monoN and monoC TF on erythropoiesis are relevant not only in steady-state, but also in the ineffective erythropoiesis that is characteristic of β-thalassemia. Because both monoN and monoC TF forms can deliver only one iron atom per TF-TFR1 binding event, our findings suggest that the improvements observed only in the Hbb th3/+Tf monoN mice were not due to iron restriction alone. We are now elucidating the mechanisms by which the two TF lobes exert their differential effects on ineffective erythropoiesis and exploring the translational potential of obligate monoN TF in the treatment of β-thalassemia. Figure 1 Figure 1. Disclosures Rivella: Ionis Pharmaceuticals: Consultancy; Meira GTx: Consultancy.

Constitutive Expression of Hypoxia Inducible Factor Downregulates the Anticoagulant Protein S in a Unique Chuvash Polycythemia Population with Erythrocytosis

Background: Chuvash polycythemia is a hematological disorder present worldwide but endemic to the Chuvash population, a Turkish ethnic group in Russia. The disorder is caused by a homozygous germline mutation (R200W) in the von Hippel Lindau gene (1,2). This mutation impairs binding of pVHL to hypoxia-inducible factor 1-alpha (HIF-1a); lack of this interaction prevents degradation of HIF-1a (2,3). The resultant upregulation of HIF-1a, even in a normal oxygen state, increases the activity of erythropoietin, thereby causing polycythemia (2,3). Affected individuals experience increased rates of arterial and venous thrombosis unrelated to the increased concentration of hemoglobin (4,5). Aims: To determine whether upregulation of HIF-1a in Chuvash polycythemia individuals causes a decreased level of the antithrombotic Protein S. A decreased level of Protein S may explain the increased risks of thrombotic events and vascular abnormalities in the Chuvash population. Methods: Enzyme-linked immunosorbent assays (ELISA) were performed to measure total Protein S concentration in Chuvash and control plasma. Immunoblotting was performed to confirm the ELISA measurements. Additional assays to measure free Protein S and thrombin generation assays with and without Protein S will be performed to determine whether Chuvash plasma has low free Protein S and whether Protein S supplementation will improve the thrombotic phenotypes. Results: Total Protein S concentration measured by ELISA was lower in Chuvash individuals compared with controls. In addition, we have completed immunoblotting of seven Chuvash samples and three control samples. Our results indicated that Chuvash plasma had lower amounts of Protein S compared with the controls. Immunoblotting of additional Chuvash samples is in progress, and we are planning to measure the free Protein S in Chuvash samples. Conclusion: Our preliminary results suggest a decreased level of total Protein S in individuals with Chuvash polycythemia. Because an increased hemoglobin concentration does not increase the rates of thromboembolic events in this population, our finding of a reduced amount of anticoagulant Protein S may explain the hypercoagulability that Chuvash individuals experience. References Gordeuk, V.R., et al., Chuvash polycythemia VHLR200W mutation is associated with down-regulation of hepcidin expression. Blood, 2011. 118(19): p. 5278-82. Gordeuk, V.R., et al., Congenital disorder of oxygen sensing: association of the homozygous Chuvash polycythemia VHL mutation with thrombosis and vascular abnormalities but not tumors. Blood, 2004. 103(10): p. 3924-32. Gordeuk, V.R. and J.T. Prchal, Vascular complications in Chuvash polycythemia. Semin Thromb Hemost, 2006. 32(3): p. 289-94. Sergueeva, A., et al., Prospective study of thrombosis and thrombospondin-1 expression in Chuvash polycythemia. Haematologica, 2017. 102(5): p. e166-e169. Gordeuk, V.R., N.S. Key, and J.T. Prchal, Re-evaluation of hematocrit as a determinant of thrombotic risk in erythrocytosis. Haematologica, 2019. 104(4): p. 653-658. Disclosures Gordeuk: Modus Therapeutics: Consultancy; Novartis: Research Funding; Incyte: Research Funding; Emmaus: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; CSL Behring: Consultancy.

A Monoclonal Antibody Targeting ALK2 As a Potential Therapeutic Agent for Anemia of Inflammation

Background: Iron homeostasis is primarily regulated by hepcidin, a hormone predominantly expressed in the liver. Hepcidin activates the degradation of the transmembrane iron exporter ferroportin, thereby downregulating the release of iron from cells. Hepcidin expression is, at least partly, regulated in response to signaling of the type I TGF-β receptor ALK2, via SMAD2/3 phosphorylation. IL-6, which is commonly elevated in chronic kidney disease (CKD) and other inflammatory conditions, upregulates hepcidin expression and reduces serum iron bioavailability. As a result, chronic inflammatory conditions are often accompanied by secondary anemia of inflammation (AI). We have previously demonstrated that ALK2 inhibition suppressed hepcidin expression in rodents, monkeys and healthy humans. We further described that administration of a selective small molecule ALK2 kinase inhibitor (KTI-2338) reversed changes in hepcidin and iron in a mouse model of CKD, supporting the potential benefit of ALK2 inhibition in AI. Another approach to targeting ALK2 signaling is use of a neutralizing antibody. KTI-018 is a neutralizing ALK2 antibody with high affinity and selectivity for ALK2. This biologic has been demonstrated to reduce serum hepcidin and increase serum iron in healthy non-human primates. Aims: To further elucidate the specific contribution of ALK2 signaling as a driver in AI, and to determine the therapeutic potential of the antibody in this type of anemia, we assessed the effect of KTI-018 in the CKD mouse model. Methods: The study was conducted with 6-week-old male C57Bl/6 mice. Mice in the CKD cohort (CKD) were treated with once daily oral administration of adenine, a compound that metabolizes to 2,8-dihydroxyadenine, forming crystals in the proximal tubular epithelia and causing inflammation and fibrosis in the kidneys. Mice in the control cohort (healthy) received once daily oral administration of vehicle. Upon confirmation of disease, the CKD cohort was subdivided into two groups. The treatment group received twice weekly intraperitoneal treatment with KTI-018 (CKD-KTI-018), and the control group received tris-buffered saline (CKD-TBS). Healthy mice received TBS only. All mice were maintained on their assigned daily adenine or vehicle regimen. At day 53, the study was terminated and hematologic parameters, serum hepcidin, iron, and IL-6 levels were assessed. Results: After 42 days of adenine or vehicle administration, serum hepcidin, serum iron, and hematologic parameters were assessed in representative cohorts of CKD-TBS and healthy mice. The CKD-TBS cohort experienced changes associated with anemia of inflammation as compared to the healthy mice, including increased hepcidin, decreased serum iron, and decreased hematologic parameters. The differences between the healthy and CKD-TBS groups were maintained through the duration of the study. At study termination, CKD-TBS mice had increased serum IL-6 levels (218%), elevated serum hepcidin (149%), and reduced serum iron (-30%) as compared to the healthy mice. Laboratory findings characteristic of anemia were present in the CKD-TBS group, including decreased red blood cells (-6.1%), hemoglobin (-13.2%), and reticulocyte hemoglobin content (-9.3%) as compared to healthy mice. In contrast, CKD-KTI-018 mice had decreased serum hepcidin (-25%) and increased serum iron (59%) as compared to CKD-TBS mice. This restoration of serum iron corresponded to improvements in red blood cells, hemoglobin, and reticulocyte hemoglobin content, which were increased by 7.6%, 9.6%, and 6.7%, respectively, in the CKD-KTI-018 mice as compared to the CKD-TBS mice. These results demonstrate that, by decreasing serum hepcidin, KTI-018 increased the bioavailability of iron, which led to the restoration of hematologic parameters and appeared to reverse AI in mice. Discussion: In this study, a neutralizing ALK2 antibody decreased serum hepcidin, increased serum iron and consequently reversed AI in a mouse model of CKD. These results support the role of ALK2 signaling in AI and suggest that inhibition of ALK2 may be a potential treatment approach for anemia resulting from CKD and other chronic inflammatory diseases. Future studies will explore if ALK2 inhibition may prevent or treat progression of CKD itself, and the role that ALK2 inhibition may play in other chronic inflammatory conditions associated with elevated hepcidin. Disclosures Medeiros: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Backus: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Materna: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Fisher: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lachey: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Seehra: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Non-Pharmacological Interventions for Anemia Treatment: Systematic Review

The global prevalence of anemia in non-pregnant women, of childbearing age, is estimated at 29.0% and is more common in low- and middle-income countries, women belonging to low socioeconomic strata. iron deficiency can cause direct or risky disability. To determine the effectiveness of Non-Pharmacological Interventions for the treatment of anemia. The method used is with the help of electronic databases from journals that have been published through PubMed, Proquest, EBSCOhost, and Science Direct as many as 6 articles were reviewed from 1186 articles. The 6 articles reviewed in this study with varied respondents using patients, male rats, nurses, Sprague-Dawley (SD) Rattus norvegicus Domestica, bovine serum, premature neonates. Non-pharmacological interventions developed in the treatment of anemia in both human and animal samples as well as the development of treatment and laboratory examinations in the treatment of anemia are Nursing Delirium Screening scale, Hepcidin expression, pain identification, HM10760A, Divalent metal transporter DMT1/SLC11A2, conservative management.

Hyperphosphatemia increases inflammation to exacerbate anemia and skeletal muscle wasting independently of FGF23-FGFR4 signaling

Elevations in plasma phosphate concentrations (hyperphosphatemia) occur in chronic kidney disease (CKD), in certain genetic disorders, and following the intake of a phosphate-rich diet. Whether hyperphosphatemia and/or associated changes in metabolic regulators, including elevations of fibroblast growth factor 23 (FGF23) directly contribute to specific complications of CKD is uncertain. Here we report that similar to patients with CKD, mice with adenine-induced CKD develop inflammation, anemia and skeletal muscle wasting. These complications are also observed in mice fed high phosphate diet even without CKD. Ablation of pathologic FGF23-FGFR4 signaling did not protect mice on an increased phosphate diet or mice with adenine-induced CKD from these sequelae. However, low phosphate diet ameliorated anemia and skeletal muscle wasting in a genetic mouse model of CKD. Our mechanistic in vitro studies indicate that phosphate elevations induce inflammatory signaling and increase hepcidin expression in hepatocytes, a potential causative link between hyperphosphatemia, anemia and skeletal muscle dysfunction. Our study suggests that high phosphate intake, as caused by the consumption of processed food, may have harmful effects irrespective of pre-existing kidney injury, supporting not only the clinical utility of treating hyperphosphatemia in CKD patients but also arguing for limiting phosphate intake in healthy individuals.