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