Effect of Inducible Antioxidants on Hemoglobin Toxicity

Summary

Principal Investigator: Raymond Regan
Abstract: DESCRIPTION (provided by applicant): Hemorrhage accompanies most significant brain trauma, and is the primary event in about 15% of strokes. A growing body of evidence suggests that hemoglobin (Hb) release from lysing erythrocytes may contribute to oxidative injury to tissue surrounding a hematoma. Prior studies have demonstrated that Hb toxicity is mediated by transfer of its heme moieties to nearby cells, which is favored by its oxidation to methemoglobin. Hemin, the oxidized form of heme, is then catabolized by the heme oxygenase enzymes to iron, carbon monoxide, and biliverdin. The cytoprotective effects of the latter two products may be overwhelmed if iron sequestration is inadequate. However, the iron detoxification strategies of neurons and astrocytes remain largely undefined. Preliminary experiments suggest that neurons export extracellular iron, where it must be sequestered to prevent oxidative membrane injury;astrocytes induce ferritin, consistent with intracellular storage. The goal of this project is to define the role of ferritin and transferrin in mitigating heme-mediated injury to neurons and astrocytes, and to devise a strategy to optimize this process. Our experimental aims are as follows: 1) Specifically increase expression of H or L-ferritin subunits in cultured neurons and astrocytes by adenoviral transfer of their genes. Alternatively, culture astrocytes and neurons from iron regulatory protein (IRP) knockout mice, which have markedly increased cell ferritin. Determine the effect of ferritin gene transfer or IRP knockdown on culture reactive oxygen species (ROS) formation, protein oxidation, and cell viability after Hb or hemin exposure. 2) Treat cultured neurons and astrocytes with [55Fe]hemin. Quantify subsequent 55Fe release into the culture medium or deposited in cell ferritin. Assess the effect of apotransferrin and holotransferrin on cell vulnerability to heme-mediated injury. 3) Stereo- tactically inject blood into the striata of hypotransferrinemic mice or non-mutant littermates. Alternatively, increase ferritin expression in blood-injected mice by adenoviral transfer of ferritin genes or shRNA to IRP's. At 72 and 144 hours, determine the effect on cell viability, protein and lipid oxidation, and axonal injury in surrounding tissue. The information gained in this project may lead to new treatments for victims of hemorrhagic stroke and head trauma. The ultimate goal is to reduce brain injury in tissue surrounding a blood clot, and to thereby improve the likelihood of survival and return to an independent, productive life.
Funding Period: ----------------2001 - ---------------2011-
more information: NIH RePORT

Top Publications

  1. ncbi Inhibition of the ERK/MAP kinase pathway attenuates heme oxygenase-1 expression and heme-mediated neuronal injury
    Luna Benvenisti-Zarom
    Department of Emergency Medicine, Thomas Jefferson University, 1020 Sansom Street, Thompson Building, Room 239, Philadelphia, PA 19107, United States
    Neurosci Lett 398:230-4. 2006
  2. pmc Effect of iron chelators on methemoglobin and thrombin preconditioning
    Jing Chen-Roetling
    Department of Emergency Medicine, Thomas Jefferson University, 1025 Walnut Street, College Building Room 813, Philadelphia, PA 19107, USA
    Transl Stroke Res 3:452-9. 2012
  3. pmc Iron regulatory proteins increase neuronal vulnerability to hydrogen peroxide
    Raymond F Regan
    Department of Emergency Medicine, Thomas Jefferson University, 1020 Sansom Street, Thompson 239, Philadelphia, PA 19107, USA
    Biochem Biophys Res Commun 375:6-10. 2008
  4. pmc Neurons lacking iron regulatory protein-2 are highly resistant to the toxicity of hemoglobin
    Raymond F Regan
    Department of Emergency Medicine, Thomas Jefferson University, 1020 Sansom Street, Thompson Building Room 239, Philadelphia, PA 19107, USA
    Neurobiol Dis 31:242-9. 2008
  5. ncbi Time course of increased heme oxygenase activity and expression after experimental intracerebral hemorrhage: correlation with oxidative injury
    Mai Chen
    Department of Emergency Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania, USA
    J Neurochem 103:2015-21. 2007
  6. ncbi Attenuation of oxidative injury after induction of experimental intracerebral hemorrhage in heme oxygenase-2 knockout mice
    Yan Qu
    Department of Emergency Medicine, Thomas Jefferson University, Philadelphia, Pennyslvania 19107, USA
    J Neurosurg 106:428-35. 2007
  7. pmc Heme oxygenase-2 is a critical determinant for execution of an acute inflammatory and reparative response
    Francesca Seta
    Department of Pharmacology, New York Medical College, Grassland Reservation, Valhalla, NY 10595, USA
    Am J Pathol 169:1612-23. 2006
  8. pmc Effect of heme oxygenase-1 on the vulnerability of astrocytes and neurons to hemoglobin
    Jing Chen-Roetling
    Thomas Jefferson University, 1020 Sansom Street, Thompson 239, Philadelphia, PA 19107, USA
    Biochem Biophys Res Commun 350:233-7. 2006
  9. pmc Hemopexin decreases hemin accumulation and catabolism by neural cells
    Jing Chen-Roetling
    Department of Emergency Medicine, Thomas Jefferson University, 1025 Walnut Street, College Building Room 813, Philadelphia, PA 19107, USA
    Neurochem Int 60:488-94. 2012
  10. pmc Iron accumulation and neurotoxicity in cortical cultures treated with holotransferrin
    Jing Chen-Roetling
    Department of Emergency Medicine, Thomas Jefferson University, Philadelphia, PA 19107, USA
    Free Radic Biol Med 51:1966-74. 2011

Detail Information

Publications16

  1. ncbi Inhibition of the ERK/MAP kinase pathway attenuates heme oxygenase-1 expression and heme-mediated neuronal injury
    Luna Benvenisti-Zarom
    Department of Emergency Medicine, Thomas Jefferson University, 1020 Sansom Street, Thompson Building, Room 239, Philadelphia, PA 19107, United States
    Neurosci Lett 398:230-4. 2006
    ..These results suggest that targeting the ERK pathway may prevent HO-1 induction in response to hemin, and reduce neuronal injury...
  2. pmc Effect of iron chelators on methemoglobin and thrombin preconditioning
    Jing Chen-Roetling
    Department of Emergency Medicine, Thomas Jefferson University, 1025 Walnut Street, College Building Room 813, Philadelphia, PA 19107, USA
    Transl Stroke Res 3:452-9. 2012
    ..These results suggest a potentially deleterious effect of continuous iron chelator therapy after ICH. Intermittent therapy may remove peri-hematomal iron without negating the benefits of exposure to low concentrations of Hb or thrombin. ..
  3. pmc Iron regulatory proteins increase neuronal vulnerability to hydrogen peroxide
    Raymond F Regan
    Department of Emergency Medicine, Thomas Jefferson University, 1020 Sansom Street, Thompson 239, Philadelphia, PA 19107, USA
    Biochem Biophys Res Commun 375:6-10. 2008
    ..These results suggest that iron regulatory proteins, particularly IRP2, increase neuronal vulnerability to oxidative injury. Therapies targeting IRP2 binding to ferritin mRNA may attenuate neuronal loss due to oxidative stress...
  4. pmc Neurons lacking iron regulatory protein-2 are highly resistant to the toxicity of hemoglobin
    Raymond F Regan
    Department of Emergency Medicine, Thomas Jefferson University, 1020 Sansom Street, Thompson Building Room 239, Philadelphia, PA 19107, USA
    Neurobiol Dis 31:242-9. 2008
    ..Therapeutic strategies that target this regulatory mechanism may be beneficial after hemorrhagic CNS injuries...
  5. ncbi Time course of increased heme oxygenase activity and expression after experimental intracerebral hemorrhage: correlation with oxidative injury
    Mai Chen
    Department of Emergency Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania, USA
    J Neurochem 103:2015-21. 2007
    ..As HO-1 expression is temporally associated with increased tissue heme and increased protein carbonylation, it may be more useful as a marker of heme-mediated oxidative stress in ICH models, rather than as an index of HO activity...
  6. ncbi Attenuation of oxidative injury after induction of experimental intracerebral hemorrhage in heme oxygenase-2 knockout mice
    Yan Qu
    Department of Emergency Medicine, Thomas Jefferson University, Philadelphia, Pennyslvania 19107, USA
    J Neurosurg 106:428-35. 2007
    ..Hemoglobin breakdown is catalyzed in part by the heme oxygenase (HO) enzymes. In the present study, the authors tested the hypothesis that HO-2 gene deletion is cytoprotective in an experimental ICH model...
  7. pmc Heme oxygenase-2 is a critical determinant for execution of an acute inflammatory and reparative response
    Francesca Seta
    Department of Pharmacology, New York Medical College, Grassland Reservation, Valhalla, NY 10595, USA
    Am J Pathol 169:1612-23. 2006
    ..Thus, HO-2 sets in place a basal tone of anti-inflammatory signals that may be a prerequisite for the ordered execution of an inflammatory and reparative response...
  8. pmc Effect of heme oxygenase-1 on the vulnerability of astrocytes and neurons to hemoglobin
    Jing Chen-Roetling
    Thomas Jefferson University, 1020 Sansom Street, Thompson 239, Philadelphia, PA 19107, USA
    Biochem Biophys Res Commun 350:233-7. 2006
    ..These results suggest that HO-1 induction protects astrocytes from the oxidative toxicity of Hb, but has no effect on neuronal injury...
  9. pmc Hemopexin decreases hemin accumulation and catabolism by neural cells
    Jing Chen-Roetling
    Department of Emergency Medicine, Thomas Jefferson University, 1025 Walnut Street, College Building Room 813, Philadelphia, PA 19107, USA
    Neurochem Int 60:488-94. 2012
    ..Its beneficial effect in stroke models is unlikely to be mediated by increased production of cytoprotective heme breakdown products...
  10. pmc Iron accumulation and neurotoxicity in cortical cultures treated with holotransferrin
    Jing Chen-Roetling
    Department of Emergency Medicine, Thomas Jefferson University, Philadelphia, PA 19107, USA
    Free Radic Biol Med 51:1966-74. 2011
    ..Chelator therapy may be beneficial for acute CNS injuries associated with loss of blood-brain barrier integrity...
  11. pmc Increased striatal injury and behavioral deficits after intracerebral hemorrhage in hemopexin knockout mice
    Lifen Chen
    Department of Emergency Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA
    J Neurosurg 114:1159-67. 2011
    ..In the present study, the authors tested the hypothesis that hemopexin knockout mice would sustain more injury after experimental ICH than their wild-type counterparts...
  12. pmc Apotransferrin protects cortical neurons from hemoglobin toxicity
    Jing Chen-Roetling
    Department of Emergency Medicine, Thomas Jefferson University, 1025 Walnut Street, College Building Room 813, Philadelphia, PA 19107, USA
    Neuropharmacology 60:423-31. 2011
    ..These results suggest that apotransferrin may mitigate the neurotoxicity of hemoglobin after intracerebral hemorrhage. Increasing its concentration in perihematomal tissue may be beneficial...
  13. pmc Accelerated hemolysis and neurotoxicity in neuron-glia-blood clot co-cultures
    Kellie M Jaremko
    Department of Emergency Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA
    J Neurochem 114:1063-73. 2010
    ..These results suggest that hemoglobin release from clotted blood is accelerated by adjacent neurons and glia. Subsequent neurotoxicity is mediated by both iron-dependent and excitotoxic injury pathways...
  14. pmc Iron regulatory protein-2 knockout increases perihematomal ferritin expression and cell viability after intracerebral hemorrhage
    Mai Chen
    Department of Emergency Medicine, Thomas Jefferson University, 1020 Sansom Street, Philadelphia, PA 19107, USA
    Brain Res 1337:95-103. 2010
    ..IRP2 binding activity may be a novel therapeutic target after hemorrhagic CNS injuries...
  15. pmc Increasing expression of H- or L-ferritin protects cortical astrocytes from hemin toxicity
    Zhi Li
    Department of Emergency Medicine, Thomas Jefferson University, Philadelphia, PA 19107, USA
    Free Radic Res 43:613-21. 2009
    ..These results suggest that over-expression of either H- or L-ferritin protects astrocytes from hemin and may be beneficial after CNS haemorrhage...
  16. pmc Minocycline attenuates iron neurotoxicity in cortical cell cultures
    Jing Chen-Roetling
    Department of Emergency Medicine, Thomas Jefferson University, Philadelphia, PA 19107, USA
    Biochem Biophys Res Commun 386:322-6. 2009
    ..These results suggest that attenuation of iron neurotoxicity may contribute to the beneficial effect of minocycline in hemorrhagic stroke and other CNS injury models...