free heme triggers weibel palade body exocytosis and vaso-occlusion in sickle mice
TRANSCRIPT
John Belcherand
Greg Vercellotti
Free Heme Triggers Weibel Palade Body Exocytosis and Vaso-occlusion in
Sickle Mice
Disclosure
Sangart research funding and consultant
Heme Structure
Heme (Fe2+), Hemin (Fe3+/3Cl-), Hematin (Fe3+/3OH-)
Hemolysis Promotes Endothelial Cell Activation
Haptoglobin
NO
eNOS
Met-Hb
HEMIN
Hemopexin
H2O2
Fe3+
ROS
Fe2+
ROS
Endothelial Cell ActivationTLR4
TLR4
Oxidative Stress, Inflammation and Vaso-occlusion: A Vicious Cycle Fueled by Hemolysis
ReactiveOxygenSpecies
RBC & LeukocyteAdhesion
Endothelial Cell Adhesion Molecule Expression
NF-kBActivation
Vaso-occlusion& Ischemia
Sickle RBCHemolysis
Activated Endothelium
Hb
HeminMetHb
Hemin MetHb Hb ActivatedLeukocytes
THO-1?
THO-1?
Reperfusion
XDH XO
Why HO-1?
Hypothesis:HO-1 inhibits vaso-occlusion in
transgenic sickle mice in response to hypoxia-
reoxygenation.
• 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
Murine Model of Vaso-occlusion
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.
HO-1 Induction Inhibits Hypoxia-induced Vascular Stasis in Transgenic Sickle Mice
HMOX1 Gene Transfer using aSleeping Beauty Transposase with an Albumin
Promoter Increases Hepatic HO-1 Activity 3-Fold in Sickle Mice
0
50
100
150
Control LRS wt-HO-1 ns-HO-1 Hemin
Bili
rub
in (
pm
ol/m
g p
rote
in) **
*
HO-1 Overexpression in the Liver of Sickle MiceInhibits Hypoxia-induced Vascular Stasis in the Skin
0
5
10
15
20
25
Control LRS wt-HO-1 ns-HO-1 Hemin
Me
an
% S
tas
is
*
*
Oxidative Stress, Inflammation and Vaso-occlusion: A Vicious Cycle Inhibited by HO-1
ReactiveOxygenSpecies
RBC & LeukocyteAdhesion
Endothelial Cell Adhesion Molecule Expression
NF-kBActivation
Vaso-occlusion& Ischemia
Sickle RBCHemolysis
Activated Endothelium
Hb
HeminMetHb
Hemin MetHb Hb ActivatedLeukocytes
THO-1
THO-1
Reperfusion
XDH XO XX
Normal Endothelium
Can plasma hemoglobin and hemin induce vascular stasis
in sickle mice without hypoxia-reoxygenation?
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
Experimental Outline
Hemoglobin and Hemin Infusion Do Not Trigger Vascular Stasis in C57
Mice
0%
10%
20%
30%
40%
50%
0 1 2 3 4
Stas
is (%
)
Time After Infusion (hours)
Saline
HbA
Hemin
0%
10%
20%
30%
40%
50%
0 1 2 3 4
Stas
is (%
)
Time After Infusion (hours)
Saline
0%
10%
20%
30%
40%
50%
0 1 2 3 4
Stas
is (%
)
Time After Infusion (hours)
Saline
HbA
0%
10%
20%
30%
40%
50%
0 1 2 3 4
Stas
is (%
)
Time After Infusion (hours)
Saline
HbA
Hemin
0%
10%
20%
30%
40%
50%
0 1 2 3 4
Stas
is (%
)
Time After Infusion (hours)
Saline
HbA
Hemin
Water
Hemoglobin, Hemin and Water Infusion Trigger Vascular Stasis in
Sickle Mice
Hemoglobin-induced Vascular Stasis
is Dose Dependent
0%
10%
20%
30%
40%
50%
0 1 2 3 4
Stas
is (%
)
Time After Infusion (hours)
HbA 8.0 nmol/g
HbA 0.8nmol/g
HbA 0.08 nmol/g
Saline
Hemin Induced Vascular Stasis is Dose Dependent
0%
10%
20%
30%
40%
50%
0 1 2 3 4
Stas
is (%
)
Time After Infusion (hours)
Hemin 32 nmols/g
Hemin 3.2 nmols/g
Hemin 0.32 nmols/g
Saline
Hemin Induced Vascular Stasis: Dose Response Curve
Does hemoglobin induce vascular stasis directly or
does hemin need to be released by methemoglobin?
Methemoglobin, but not Cyanomethemoglobin, Induces Vascular Stasis in Sickle Mice
Can Haptoglobin and Hemopexin Prevent Stasis?
Haptoglobin
NO
eNOS
Met-Hb
HEMIN
Hemopexin
Haptoglobin and Hemopexin Inhibit Hemoglobin-induced Stasis
Hemopexin Inhibits Hemin-induced Stasis
How does heme induce vascular stasis in sickle
mice?
Does P-selectin play a role?
P-selectin Blocking Antibodies Inhibit Hemin-induced Stasis in Sickle Mice
Weibel Palade Body Contents
vWFP-selectinCD63Tissue plasminogen factor
IL-8EotaxinEndothelin 1Endothelin-converting enzyme
Angiopoietin 2
Weibel-Palade Body Agonists and Antagonists
AgonistsHistamine Thrombin
Reactive oxygen species HypoxiaEpinephrine SerotoninLeukotrienes FibrinShear stress (acute) VEGFATP Complement (C5-9)ADP Oxidized LDLRadiation CeramideTrauma Sphingosine-1-phosphate
AntagonistsN-acetyl-cysteine N-ethylmaleimideLipoic Acid NO (S-nitrosylation)
Does heme induce rapid Weibel-Palade Body
exocytosis?
Hemin Triggers Rapid P-selectin and Von Willebrand Factor Expression on the Surface of Endothelial Cells
In Vitro
Hemin Infusion Triggers Rapid P-selectin Expression on Pulmonary Veins
Green = CD31; Red = P-selectin; Blue = nuclei
How does heme activate endothelial cells?
TLR4 Signal Transduction with LPS
TLR4 Inhibitor TAK-242 Inhibits Heme-induced P-Selectin and vWF Expression on
Endothelial Cells
Green = P-selectin; Red = vWF; Blue = nuclei
Proposed Model
TLR4 Inhibitor TAK-242 Inhibits Heme-induced Vascular Stasis in Transgenic Sickle
Mice
Calphostin C Inhibits Vascular Stasis in
Transgenic Sickle Mice
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.
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.
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