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

JD Belcher, C Chen, J Nguyen… - Blood, The Journal …, 2014 - ashpublications.org
JD Belcher, C Chen, J Nguyen, L Milbauer, F Abdulla, AI Alayash, A Smith, KA Nath…
Blood, The Journal of the American Society of Hematology, 2014ashpublications.org
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 …
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.
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