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

John D. Belcher, Chunsheng Chen, Julia Nguyen, Liming Milbauer, Fuad Abdulla, Abdu I. Alayash, Ann Smith, Karl A Nath, Robert P. Hebbel, Gregory M. Vercellotti

Research output: Contribution to journalArticle

235 Citations (Scopus)

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, α4βb1, 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.

Original languageEnglish (US)
Pages (from-to)377-390
Number of pages14
JournalBlood
Volume123
Issue number3
DOIs
StatePublished - Jan 16 2014

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Endothelial cells
Sickle Cell Anemia
Heme
Endothelial Cells
Chemical activation
Weibel-Palade Bodies
Lipopolysaccharides
von Willebrand Factor
Hemolysis
Hemopexin
Methemoglobin
Haptoglobins
P-Selectin
Hemoglobins
Adhesion
Leukocyte Rolling
CD31 Antigens
E-Selectin
Vascular Cell Adhesion Molecule-1
NADPH Oxidase

ASJC Scopus subject areas

  • Hematology
  • Biochemistry
  • Cell Biology
  • Immunology

Cite this

Belcher, J. D., Chen, C., Nguyen, J., Milbauer, L., Abdulla, F., Alayash, A. I., ... Vercellotti, G. M. (2014). Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease. Blood, 123(3), 377-390. https://doi.org/10.1182/blood-2013-04-495887

Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease. / Belcher, John D.; Chen, Chunsheng; Nguyen, Julia; Milbauer, Liming; Abdulla, Fuad; Alayash, Abdu I.; Smith, Ann; Nath, Karl A; Hebbel, Robert P.; Vercellotti, Gregory M.

In: Blood, Vol. 123, No. 3, 16.01.2014, p. 377-390.

Research output: Contribution to journalArticle

Belcher, JD, Chen, C, Nguyen, J, Milbauer, L, Abdulla, F, Alayash, AI, Smith, A, Nath, KA, Hebbel, RP & Vercellotti, GM 2014, 'Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease', Blood, vol. 123, no. 3, pp. 377-390. https://doi.org/10.1182/blood-2013-04-495887
Belcher, John D. ; Chen, Chunsheng ; Nguyen, Julia ; Milbauer, Liming ; Abdulla, Fuad ; Alayash, Abdu I. ; Smith, Ann ; Nath, Karl A ; Hebbel, Robert P. ; Vercellotti, Gregory M. / Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease. In: Blood. 2014 ; Vol. 123, No. 3. pp. 377-390.
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N2 - 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, α4βb1, 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.

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