Neuregulin-1 attenuates hemolysis- and ischemia induced-cerebrovascular inflammation associated with sickle cell disease

  • Christopher Chambliss
    Corresponding authors.
    Pediatrics Institute, Emory University School of Medicine, 2015 Uppergate Drive, Atlanta, GA 30322, United States

    Aflac Cancer and Blood Disorders Center, 2015 Uppergate Drive, Atlanta, GA 30322, United States

    Cardiovascular Research Institute, Morehouse School of Medicine, 720 Westview Drive SW, Atlanta, GA 30310, United States
    Search for articles by this author
  • Jonathan K. Stiles
    Corresponding authors.
    Department of Microbiology, Biochemistry, and Immunology, Morehouse School of Medicine, 720 Westview Drive SW, Atlanta, GA 30310, United States
    Search for articles by this author
  • Beatrice E. Gee
    Pediatrics Institute, Emory University School of Medicine, 2015 Uppergate Drive, Atlanta, GA 30322, United States

    Aflac Cancer and Blood Disorders Center, 2015 Uppergate Drive, Atlanta, GA 30322, United States

    Children's Healthcare of Atlanta, 35 Jesse Hill Jr Drive SE, Atlanta, GA 30303, United States

    Department of Pediatrics, Morehouse School of Medicine, 720 Westview Drive SW, Atlanta, GA 30310, United States
    Search for articles by this author


      • Mice with SCD show elevated plasma NRG-1 levels in comparison to healthy controls.
      • Experimental conditions mimicking hemolysis and ischemia increase NRG-1 production.
      • NRG-1 reduced inflammatory cytokine and adhesion molecule expression.
      • NRG-1 expression in SCD is likely a protective endogenous response.



      Individuals with sickle cell disease (SCD) are at severely heightened risk for cerebrovascular injury and acute cerebrovascular events, including ischemic and hemorrhagic stroke, potentially leading to impaired development and life-long physical and cognitive disabilities. Cerebrovascular injury specific to SCD includes inflammation caused by underlying conditions of chronic hemolysis and reduced cerebrovascular perfusion. The objectives of this study were to investigate whether expression of neuregulin-1β (NRG-1), an endogenous neuroprotective polypeptide, is increased in SCD or experimental conditions mimicking the hemolysis and ischemic conditions of SCD, and to determine if treatment with exogenous NRG-1 reduces markers of cerebrovascular inflammation.

      Materials and methods

      Plasma and brain-specific NRG-1 levels were measured in transgenic SCD mice. Endogenous NRG-1 levels and response to experimental conditions of excess heme and ischemia were measured in cultured human brain microvascular cells and astrocytes. Pre-treatment with NRG-1 was used to determine NRG-1’s ability to ameliorate resultant cerebrovascular inflammation.


      Plasma and brain-specific NRG-1 were elevated in transgenic SCD mice compared to healthy controls. Neuregulin-1 expression was significantly increased in cultured human microvascular cells and astrocytes exposed to excess heme and ischemia. Pre-treatment with NRG-1 reduced inflammatory chemokine (CXCL-1 and CXCL-10) and adhesion molecule (ICAM-1 and VCAM-1) expression and increased pro-angiogenic factors (VEGF-A) in microvascular cells and astrocytes exposed to excess heme and ischemia.


      Elevated NRG-1 in SCD is likely a protective endogenous response to ongoing cerebrovascular insults caused by chronic hemolysis and reduced cerebrovascular perfusion. Administration of NRG-1 to reduce cerebrovascular inflammation may be therapeutically beneficial in SCD and warrants continued investigation.



      AA (Hemoglobin AA), AB (Antibody), AC (Hemoglobin AC), AKT (Protein kinase B), ARC (Absolute reticulocyte count), AS (Hemoglobin AS), BDNF (Brain derived neurotrophic factor), CT (Cycle of threshold), CXCL-1 (C-X-C motif chemokine ligand 1), CXCL-10 (C-X-C motif chemokine ligand 10), DAPI (4,6-diamidino-2-phenylindole), DMSO (Dimethyl sulfoxide), ELISA (Enzyme-linked immunosorbent assay), ERBB4 (Erythroblastic oncogene B receptor tyrosine kinase 4), eNOS (Endothelial derived nitric oxide synthase), GFAP (Glial fibrillary acidic protein), HB (Hemoglobin), HBMVEC (Human brain microvascular endothelial cells), HO-1 (Heme oxygenase 1), GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), GLU (Glutamine), ICAM-1 (Intracellular adhesion molecule 1), IQR (Interquartile range), LYS (Lysine), MCAO (Middle cerebral artery occlusion), N/A (Not applicable), NEUN (Neuronal nuclei), NRG-1 (Neuregulin-1), PBS (Phosphate buffered saline), PDGF-AA (Platelet derived growth factor type-aa), PGK-1 (Phosphoglycerate kinase 1), RBC (Red blood cell), RPM (Rotations per minute), SCA (Sickle cell anemia), SCD (Sickle cell disease), SS (Hemoglobin SS), STAT (Signal transducer and activator of transcription proteins), TCD (Transcranial Doppler), VAL (Valine), VEGFA (Vascular endothelial growth factor a), VCAM-1 (Vascular cell adhesion molecule 1), WBC (White blood cell)
      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Journal of Stroke and Cerebrovascular Diseases
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Sundd P.
        • Gladwin M.T.
        • Novelli E.M.
        Pathophysiology of sickle cell disease.
        Annu Rev Pathol. 2018;
        • Verduzco L.A.
        • Nathan D.G.
        Sickle cell disease and stroke.
        Blood. 2009; 114: 5117-5125
        • Bernaudin F.
        • et al.
        Impact of early transcranial Doppler screening and intensive therapy on cerebral vasculopathy outcome in a newborn sickle cell anemia cohort.
        Blood. 2011; 117 (quiz 1436): 1130-1140
        • Hulbert M.L.
        • et al.
        Silent cerebral infarcts occur despite regular blood transfusion therapy after first strokes in children with sickle cell disease.
        Blood. 2011; 117: 772-779
        • Marano M.
        • et al.
        Recurrent large volume silent strokes in sickle cell disease.
        J Stroke Cerebrovasc Dis. 2014; 23: e453-e455
        • Schatz J.
        • et al.
        Poor school and cognitive functioning with silent cerebral infarcts and sickle cell disease.
        Neurology. 2001; 56: 1109-1111
        • Belcher J.D.
        • et al.
        Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease.
        Blood. 2014; 123: 377-390
        • Kassim A.A.
        • DeBaun M.R.
        Sickle cell disease, vasculopathy, and therapeutics.
        Annu Rev Med. 2013; 64: 451-466
        • Ohene-Frempong K.
        • et al.
        Cerebrovascular accidents in sickle cell disease: rates and risk factors.
        Blood. 1998; 91: 288-294
        • Uzunova V.V.
        • et al.
        Free Heme and the Polymerization of Sickle Cell Hemoglobin.
        Biophys J. 2010; 99: 1976-1985
        • Nath K.A.
        • et al.
        Role of TLR4 signaling in the nephrotoxicity of heme and heme proteins.
        Am J Physiol Renal Physiol. 2018; 314: F906-f914
        • Vendrame F.
        • et al.
        Differences in heme and hemopexin content in lipoproteins from patients with sickle cell disease.
        J Clin Lipidol. 2018; 12: 1532-1538
        • Ansari J.
        • Gavins F.N.E.
        Ischemia-reperfusion injury in sickle cell disease: from basics to therapeutics.
        Am J Pathol. 2019; 189: 706-718
        • Henry E.R.
        • et al.
        Treatment of sickle cell disease by increasing oxygen affinity of hemoglobin.
        Blood. 2021;
        • Uchida K.
        • et al.
        Effect of erythrocytapheresis on arterial oxygen saturation and hemoglobin oxygen affinity in patients with sickle cell disease.
        Am J Hematol. 1998; 59: 5-8
        • Vichinsky E.P.
        • et al.
        Causes and outcomes of the acute chest syndrome in sickle cell disease. National Acute Chest Syndrome Study Group.
        N Engl J Med. 2000; 342: 1855-1865
        • Birchmeier C.
        • Bennett D.L.
        Neuregulin/ErbB signaling in developmental myelin formation and nerve repair.
        Curr Top Dev Biol. 2016; 116: 45-64
        • Croslan D.J.R.
        • et al.
        Neuroprotective effects of Neuregulin-1 on B35 Neuronal Cells following Ischemia.
        Brain Res. 2008; 1210: 39-47
        • Mei L.
        • Nave K.A.
        Neuregulin-ERBB signaling in the nervous system and neuropsychiatric diseases.
        Neuron. 2014; 83: 27-49
        • Simmons L.J.
        • et al.
        Regulation of inflammatory responses by neuregulin-1 in brain ischemia and microglial cells in vitro involves the NF-kappa B pathway.
        J Neuroinflammation. 2016; 13: 237
        • Hedhli N.
        • et al.
        Endothelial-Derived Neuregulin Protects the Heart against Ischemic Injury.
        Circulation. 2011; 123: 2254-2262
        • Hedhli N.
        • Kalinowski A.
        • K S.R.
        Cardiovascular effects of neuregulin-1/ErbB signaling: role in vascular signaling and angiogenesis.
        Curr Pharm Des. 2014; 20: 4899-4905
        • Liu M.
        • et al.
        Neuregulin-1 attenuates experimental cerebral malaria (ECM) pathogenesis by regulating ErbB4/AKT/STAT3 signaling.
        J Neuroinflamm. 2018; 15: 104
        • Mòdol-Caballero G.
        • et al.
        Neuregulin 1 reduces motoneuron cell death and promotes neurite growth in an in vitro model of motoneuron degeneration.
        Front Cell Neurosci. 2017; 11: 431
        • Adams R.J.
        • et al.
        Prevention of a first stroke by transfusions in children with sickle cell anemia and abnormal results on transcranial Doppler ultrasonography.
        N Engl J Med. 1998; 339: 5-11
        • Liu M.
        • et al.
        Heme mediated STAT3 activation in severe malaria.
        PLoS One. 2012; 7: e34280
        • Solomon W.
        • et al.
        Neuregulin-1 attenuates mortality associated with experimental cerebral malaria.
        J Neuroinflammation. 2014; 11: 9
        • Rosler T.W.
        • et al.
        Biodistribution and brain permeability of the extracellular domain of neuregulin-1-beta1.
        Neuropharmacology. 2011; 61: 1413-1418
        • Surles-Zeigler M.C.
        • et al.
        Transcriptomic analysis of neuregulin-1 regulated genes following ischemic stroke by computational identification of promoter binding sites: A role for the ETS-1 transcription factor.
        PLoS One. 2018; 13e0197092
        • Xu Z.
        • et al.
        Neuroprotection by neuregulin-1 following focal stroke is associated with the attenuation of ischemia-induced pro-inflammatory and stress gene expression.
        Neurobiol Dis. 2005; 19: 461-470
        • Hyacinth H.I.
        • et al.
        Plasma BDNF and PDGF-AA levels are associated with high TCD velocity and stroke in children with sickle cell anemia.
        Cytokine. 2012; 60: 302-308
        • Chambliss C.
        • et al.
        Elevated neuregulin-1β levels correlate with plasma biomarkers of cerebral injury and high stroke risk in children with sickle cell anemia.
        Endocr Metab Sci. 2021; 3100088
        • Kato G.J.
        • Steinberg M.H.
        • Gladwin M.T.
        Intravascular hemolysis and the pathophysiology of sickle cell disease.
        J Clin Investig. 2017; 127: 750-760
        • Ofori-Acquah S.F.
        • et al.
        Elevated circulating angiogenic progenitors and white blood cells are associated with hypoxia-inducible angiogenic growth factors in children with sickle cell disease.
        Anemia. 2012; 2012156598
        • Wu L.C.
        • et al.
        Correction of sickle cell disease by homologous recombination in embryonic stem cells.
        Blood. 2006; 108: 1183-1188
        • Harbuzariu A.
        • et al.
        Modelling heme-mediated brain injury associated with cerebral malaria in human brain cortical organoids.
        Sci Rep. 2019; 9: 19162
        • Liu J.-J.
        • et al.
        A novel method for oxygen glucose deprivation model in organotypic spinal cord slices.
        Brain Res Bull. 2017; 135: 163-169
        • Tornabene E.
        • et al.
        Effects of oxygen-glucose deprivation (OGD) on barrier properties and mRNA transcript levels of selected marker proteins in brain endothelial cells/astrocyte co-cultures.
        PLOS ONE. 2019; 14e0221103
        • Zhang B.
        • Li J.
        Phoenixin-14 protects human brain vascular endothelial cells against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced inflammation and permeability.
        Arch Biochem Biophys. 2020; 682108275
      1. National Research Council Guide for the Care and Use of Laboratory Animals.
        8th ed. National Academy Press, Washington, DC2010
        • Parasuraman S.
        • Raveendran R.
        • Kesavan R.
        Blood sample collection in small laboratory animals.
        J Pharmacol Pharmacother. 2010; 1: 87-93
        • Fraser S.T.
        • et al.
        Heme oxygenase-1: a critical link between iron metabolism, erythropoiesis, and development.
        Adv Hematol. 2011; 2011473709
        • D'Alessandro S.
        • et al.
        Effect of hypoxia on gene expression in cell populations involved in wound healing.
        Biomed Res Int. 2019; 20192626374
        • Mense S.M.
        • et al.
        Gene expression profiling reveals the profound upregulation of hypoxia-responsive genes in primary human astrocytes.
        Physiol Genomics. 2006; 25: 435-449
        • Michiels C.
        • Arnould T.
        • Remacle J.
        Endothelial cell responses to hypoxia: initiation of a cascade of cellular interactions.
        Biochim Biophys Acta. 2000; 1497: 1-10
        • Bussolati B.
        • Mason J.C.
        Dual role of VEGF-induced heme-oxygenase-1 in angiogenesis.
        Antioxid Redox Signal. 2006; 8: 1153-1163
        • Long J.
        • et al.
        The therapeutic effect of vascular endothelial growth factor gene- or heme oxygenase-1 gene-modified endothelial progenitor cells on neovascularization of rat hindlimb ischemia model.
        J Vasc Surg. 2013; 58 (e2): 756-765
        • Hyacinth H.I.
        • et al.
        Higher prevalence of spontaneous cerebral vasculopathy and cerebral infarcts in a mouse model of sickle cell disease.
        J Cereb Blood Flow Metab. 2017; (271678x17732275)
        • Law A.J.
        • et al.
        Neuregulin-1 (NRG-1) mRNA and protein in the adult human brain.
        Neuroscience. 2004; 127: 125-136
        • Liu X.
        • et al.
        Specific regulation of NRG1 isoform expression by neuronal activity.
        J Neurosci. 2011; 31: 8491
        • Lok J.
        • et al.
        Neuregulin-1 signaling in brain endothelial cells.
        J Cereb Blood Flow Metab. 2009; 29: 39-43
        • Thompson R.J.
        • et al.
        Comparison of neuregulin-1 expression in olfactory ensheathing cells, Schwann cells and astrocytes.
        J Neurosci Res. 2000; 61: 172-185
        • Wu L.
        • et al.
        Neuregulin1-β decreases IL-1β-induced neutrophil adhesion to human brain microvascular endothelial cells.
        Transl Stroke Res. 2015; 6: 116-124
        • Belcher J.D.
        • et al.
        Oral carbon monoxide therapy in murine sickle cell disease: beneficial effects on vaso-occlusion, inflammation and anemia.
        Plos One. 2018; 13e0205194
        • Shakeri H.
        • et al.
        Neuregulin-1 compensates for endothelial nitric oxide synthase deficiency.
        Am J Physiol Heart Circ Physiol. 2021; 320: H2416-h2428
        • Gomperts E.
        • et al.
        The role of carbon monoxide and heme oxygenase in the prevention of sickle cell disease vaso-occlusive crises.
        Am J Hematol. 2017; 92: 569-582
        • Mack A.K.
        • Kato G.J.
        Sickle cell disease and nitric oxide: a paradigm shift?.
        Int J Biochem Cell Biol. 2006; 38: 1237-1243
        • Jabbour A.
        • et al.
        Parenteral administration of recombinant human neuregulin-1 to patients with stable chronic heart failure produces favourable acute and chronic haemodynamic responses.
        Eur J Heart Fail. 2011; 13: 83-92
      2. Pittalwala, I. Hope on the horizon for treating stroke. 2019 [cited 2021 April 1st]; A stroke treatment developed by researcher Byron Ford at the University of California, Riverside, has moved toward clinical trials. ]. Available from: