Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease

Blood. 2014 Jan 16;123(3):377-90. doi: 10.1182/blood-2013-04-495887. Epub 2013 Nov 25.


Treatment of sickle cell disease (SCD) is hampered by incomplete understanding of pathways linking hemolysis to vaso-occlusion. We investigated these pathways in transgenic sickle mice. Infusion of hemoglobin or heme triggered vaso-occlusion in sickle, but not normal, mice. Methemoglobin, but not heme-stabilized cyanomethemoglobin, induced vaso-occlusion, indicating heme liberation is necessary. In corroboration, hemoglobin-induced vaso-occlusion was blocked by the methemoglobin reducing agent methylene blue, haptoglobin, or the heme-binding protein hemopexin. Untreated HbSS mice, but not HbAA mice, exhibited ∼10% vaso-occlusion in steady state that was inhibited by haptoglobin or hemopexin infusion. Antibody blockade of adhesion molecules P-selectin, von Willebrand factor (VWF), E-selectin, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, platelet endothelial cell (EC) adhesion molecule 1, α4β1, or αVβ3 integrin prevented vaso-occlusion. Heme rapidly (5 minutes) mobilized Weibel-Palade body (WPB) P-selectin and VWF onto EC and vessel wall surfaces and activated EC nuclear factor κB (NF-κB). This was mediated by TLR4 as TAK-242 blocked WPB degranulation, NF-κB activation, vaso-occlusion, leukocyte rolling/adhesion, and heme lethality. TLR4(-/-) mice transplanted with TLR4(+/+) sickle bone marrow exhibited no heme-induced vaso-occlusion. The TLR4 agonist lipopolysaccharide (LPS) activated ECs and triggered vaso-occlusion that was inhibited by TAK-242, linking hemolysis- and infection-induced vaso-occlusive crises to TLR4 signaling. Heme and LPS failed to activate VWF and NF-κB in TLR4(-/-) ECs. Anti-LPS immunoglobulin G blocked LPS-induced, but not heme-induced, vaso-occlusion, illustrating LPS-independent TLR4 signaling by heme. Inhibition of protein kinase C, NADPH oxidase, or antioxidant treatment blocked heme-mediated stasis, WPB degranulation, and oxidant production. We conclude that intravascular hemolysis in SCD releases heme that activates endothelial TLR4 signaling leading to WPB degranulation, NF-κB activation, and vaso-occlusion.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anemia, Sickle Cell / metabolism*
  • Animals
  • Bone Marrow Cells / cytology
  • Cell Adhesion
  • Endothelial Cells / metabolism*
  • Haptoglobins / metabolism
  • Heme / chemistry
  • Hemoglobins / chemistry
  • Hemolysis
  • Hemopexin / metabolism
  • Human Umbilical Vein Endothelial Cells
  • Humans
  • Inflammation
  • Lipopolysaccharides / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • NF-kappa B p50 Subunit / metabolism
  • Oxidative Stress
  • Phenotype
  • Signal Transduction*
  • Toll-Like Receptor 4 / metabolism*
  • Vasoconstriction*
  • von Willebrand Factor / metabolism


  • Haptoglobins
  • Hemoglobins
  • Lipopolysaccharides
  • NF-kappa B p50 Subunit
  • Tlr4 protein, mouse
  • Toll-Like Receptor 4
  • von Willebrand Factor
  • Nfkb1 protein, mouse
  • Heme
  • Hemopexin