Free Heme Triggers Weibel Palade Body Exocytosis and Vaso-occlusion in Sickle Mice
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1. Free heme triggers the exocytosis of Weibel-Palade bodies in endothelial cells and causes vascular occlusion in sickle cell mouse models. 2. Heme activates endothelial cells through TLR4 signaling, inducing the rapid expression of P-selectin and von Willebrand factor. 3. Inhibiting heme detoxification or TLR4 signaling prevents heme-induced vascular stasis, suggesting potential new therapeutic strategies for sickle cell disease.
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Free Heme Triggers Weibel Palade Body Exocytosis and Vaso-occlusion in Sickle Mice
- 1. Free Heme Triggers Weibel Palade Body Exocytosis and Vaso-occlusion in Sickle Mice John Belcher and Greg Vercellotti
- 2. Disclosure Sangart research funding and consultant
- 3. Heme Structure Heme (Fe2+), Hemin (Fe3+/3Cl-), Hematin (Fe3+/3OH-)
- 4. Hemolysis Promotes Endothelial Cell Activation eNOS Hemopexin NO TLR4 HEMIN ROS Met-Hb Fe3+ Fe2+ ROS Haptoglobin TLR4
- 5. Oxidative Stress, Inflammation and Vaso-occlusion: A Vicious Cycle Fueled by Hemolysis Activated Hemin MetHb Hb Sickle RBC Leukocytes T HO-1? Hemolysis MetHb Hb Hemin Reactive Oxygen HO-1? Species NF-kB Reperfusion Activation Activated Endothelium Vaso-occlusion Endothelial Cell Adhesion & Ischemia Molecule Expression RBC & Leukocyte Adhesion
- 6. Why HO-1?
- 7. Hypothesis: HO-1 inhibits vaso-occlusion in transgenic sickle mice in response to hypoxia-reoxygenation.
- 8. Murine Model of Vaso-occlusion • Normal and transgenic sickle mice implanted with dorsal skin fold chambers • Wait 3 days and select and map flowing venules at baseline • Expose mice to 1 hour of hypoxia (7% O2) followed by re-oxygenation in room air • After 1 and 4 hours, re-examine the same mapped venules, count how many vessels become static (no flow), and calculate percent stasis
- 9. Stasis occurs in the subcutaneous venules inside the dorsal skin fold chamber of transgenic sickle mice, but not normal mice after hypoxia and reoxygenation Belcher et al. JCI 2006; 116; 808-816.
- 10. HO-1 Induction Inhibits Hypoxia-induced Vascular Stasis in Transgenic Sickle Mice
- 11. HMOX1 Gene Transfer using a Sleeping Beauty Transposase with an Albumin Promoter Increases Hepatic HO-1 Activity 3-Fold in Sickle Mice 150 * ** Bilirubin (pmol/mg protein) 100 50 0 Control LRS wt-HO-1 ns-HO-1 Hemin
- 12. HO-1 Overexpression in the Liver of Sickle Mice Inhibits Hypoxia-induced Vascular Stasis in the Skin 25 20 Mean % Stasis 15 10 * 5 * 0 Control LRS wt-HO-1 ns-HO-1 Hemin
- 13. Oxidative Stress, Inflammation and Vaso-occlusion: A Vicious Cycle Inhibited by HO-1 X Activated Leukocytes Hemin T MetHb HO-1 Hb Hemin Sickle RBC Hemolysis MetHb Hb X Reactive Oxygen HO-1 Species NF-kB Reperfusion Activation Activated Endothelium Normal Endothelium Vaso-occlusion Endothelial Cell Adhesion & Ischemia Molecule Expression RBC & Leukocyte Adhesion
- 14. Can plasma hemoglobin and hemin induce vascular stasis in sickle mice without hypoxia- reoxygenation?
- 15. Experimental Outline NY1DD transgenic sickle mice and C57 normal mice implanted with dorsal skin fold chambers Wait 3 days, select and map flowing venules at baseline Infuse stroma-free hemoglobin, hemin, water to induce hemolysis, or saline After 1 and 4 hours, re-examine the same mapped venules, count how many vessels become static (no flow) and calculate percent stasis
- 16. Hemoglobin and Hemin Infusion Do Not Trigger Vascular Stasis in C57 Mice 50% 40% 30% Stasis (%) Saline HbA 20% Hemin 10% 0% 0 1 2 3 4 Time After Infusion (hours)
- 17. Hemoglobin, Hemin and Water Infusion Trigger Vascular Stasis in Sickle Mice 50% 40% Saline 30% Saline Saline Stasis (%) HbA HbA 20% Hemin Hemin Water 10% 0% 0 1 2 3 4 Time After Infusion (hours)
- 18. Hemoglobin-induced Vascular Stasis is Dose Dependent 50% HbA 8.0 nmol/g HbA 0.8nmol/g 40% HbA 0.08 nmol/g Saline 30% Stasis (%) 20% 10% 0% 0 1 2 3 4 Time After Infusion (hours)
- 19. Hemin Induced Vascular Stasis is Dose Dependent 50% Hemin 32 nmols/g Hemin 3.2 nmols/g 40% Hemin 0.32 nmols/g Saline 30% Stasis (%) 20% 10% 0% 0 1 2 3 4 Time After Infusion (hours)
- 20. Hemin Induced Vascular Stasis: Dose Response Curve
- 21. Does hemoglobin induce vascular stasis directly or does hemin need to be released by methemoglobin?
- 22. Methemoglobin, but not Cyanomethemoglobin, Induces Vascular Stasis in Sickle Mice
- 23. Can Haptoglobin and Hemopexin Prevent Stasis? eNOS Hemopexin NO HEMIN Met-Hb Haptoglobin
- 24. Haptoglobin and Hemopexin Inhibit Hemoglobin-induced Stasis
- 25. Hemopexin Inhibits Hemin-induced Stasis
- 26. How does heme induce vascular stasis in sickle mice? Does P-selectin play a role?
- 27. P-selectin Blocking Antibodies Inhibit Hemin-induced Stasis in Sickle Mice
- 28. Weibel Palade Body Contents vWF P-selectin CD63 Tissue plasminogen factor IL-8 Eotaxin Endothelin 1 Endothelin-converting enzyme Angiopoietin 2
- 29. Weibel-Palade Body Agonists and Antagonists Agonists Histamine Thrombin Reactive oxygen species Hypoxia Epinephrine Serotonin Leukotrienes Fibrin Shear stress (acute) VEGF ATP Complement (C5-9) ADP Oxidized LDL Radiation Ceramide Trauma Sphingosine-1-phosphate Antagonists N-acetyl-cysteine N-ethylmaleimide Lipoic Acid NO (S-nitrosylation)
- 30. Does heme induce rapid Weibel-Palade Body exocytosis?
- 31. Hemin Triggers Rapid P-selectin and Von Willebrand Factor Expression on the Surface of Endothelial Cells In Vitro
- 32. Hemin Infusion Triggers Rapid P-selectin Expression on Pulmonary Veins Green = CD31; Red = P-selectin; Blue = nuclei
- 33. How does heme activate endothelial cells?
- 35. TLR4 Signal Transduction with LPS
- 38. TLR4 Inhibitor TAK-242 Inhibits Heme-induced P- Selectin and vWF Expression on Endothelial Cells Green = P-selectin; Red = vWF; Blue = nuclei
- 39. Proposed Model
- 40. TLR4 Inhibitor TAK-242 Inhibits Heme-induced Vascular Stasis in Transgenic Sickle Mice
- 41. Calphostin C Inhibits Vascular Stasis in Transgenic Sickle Mice
- 42. Conclusions HO-1 upregulation in sickle mice inhibits vascular stasis induced by hypoxia-reoxygenation. Heme, released from methemoglobin, elicits vascular stasis in transgenic sickle mice, but not normal C57 mice. Hemin triggers rapid Weibel-Palade body P-selectin and von Willebrand factor exocytosis on endothelium. P-selectin is necessary for hemin-induced vascular stasis in sickle mice. TLR4 signaling plays a critical role in Weibel-Palade body exocytosis and vaso-occlusion in sickle cell disease.
- 43. Speculation Removal and detoxification of hemoglobin and heme are vital during hemolysis. Haptoglobin and hemopexin supplements or modulators should be developed for sickle cell disease. Novel therapies focusing on the consequences of endothelial cell/heme interactions such as TLR4 inhibitors or P-selectin antagonists should be considered in sickle cell disease. These observations have broader implications for sepsis, hemolytic anemia, blood transfusion and even atherosclerosis.
- 44. Acknowledgements Dr. Chunsheng Chen Julia Nguyen Carol Bruzzone Dr. Sethu Nair Paul Marker Dr. Heather Bechtel Dr. Arif Somani Dr. Abdu Alayash Dr. Ann Smith Dr. Robert Hebbel Dr. Clifford Steer Research was supported by NHLBI PO-1 HL55552
Hinweis der Redaktion
- Free heme is a tightly regualted by the body because under oxidative stress the heme ring can be opened releasing catalytic iron. Via the fenton reaction, as seen on the left, this free iron can then generage a hydroxyl radical. Therefore, as illustrated as on the right, a number of conditions such as hemorrhage, the afforementioned SCD, malaria, and other conditions leads to a cycle of hemolysis, inflammation, and oxidative stress.