title: Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease.
abstract: 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.
authors.affiliation: Division of Hematology, Oncology and Transplantation, Vascular Biology Center, University of Minnesota, Minneapolis, MN;
authors.forename: John D
authors.initials: JD
authors.lastname: Belcher
authors.forename: Chunsheng
authors.initials: C
authors.lastname: Chen
authors.forename: Julia
authors.initials: J
authors.lastname: Nguyen
authors.forename: Liming
authors.initials: L
authors.lastname: Milbauer
authors.forename: Fuad
authors.initials: F
authors.lastname: Abdulla
authors.forename: Abdu I
authors.initials: AI
authors.lastname: Alayash
authors.forename: Ann
authors.initials: A
authors.lastname: Smith
authors.forename: Karl A
authors.initials: KA
authors.lastname: Nath
authors.forename: Robert P
authors.initials: RP
authors.lastname: Hebbel
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Vercellotti
mesh_terms: D000755:Anemia, Sickle Cell / Q000378:metabolism*; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D002448:Cell Adhesion; D042783:Endothelial Cells / Q000378:metabolism*; D006242:Haptoglobins / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry; D006461:Hemolysis; D006466:Hemopexin / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D007249:Inflammation; D008070:Lipopolysaccharides / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D052002:NF-kappa B p50 Subunit / Q000378:metabolism; D018384:Oxidative Stress; D010641:Phenotype; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000378:metabolism*; D014661:Vasoconstriction; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D006242:Haptoglobins; D006454:Hemoglobins; D008070:Lipopolysaccharides; D052002:NF-kappa B p50 Subunit; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D014841:von Willebrand Factor; C073701:Nfkb1 protein, mouse; D006418:Heme; D006466:Hemopexin