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Adenosine A3 Receptor Activation Attenuates
Lung Ischemia-Reperfusion Injury
via a Neutrophil-Dependent Mechanism
Ashish K. Sharma, Daniel P. Mulloy, Lucas G. Fernandez, Vanessa Hajzus,
Heesung S. Kim, Yunge Zhao, Christine L. Lau, Victor E. Laubach and
Irving L. Kron
Department of Surgery
University of Virginia Health System
Charlottesville, VA
Presenter Disclosure
Matthew L. Stone
The following relationships exist related to this
presentation:
ASHISH K. SHARMA
No Relationships to Disclose
Lung Ischemia-Reperfusion (IR) Injury:
The clinical problem after transplantation
• Primary graft dysfunction
• 20-30% of recipients
• Increased risk of Bronchiolitis
Obliterans
Left lung transplant
IR Injury - Pathophysiology
• Restoration of blood flow:
� Oxidative stress
� Proinflammatory cytokines
� Inflammation
NORMAL
• Innate immune response:
� Macrophage
� T cell
� Neutrophils
IR
Role of Adenosine in Inflammation
• Adenosine
� Increased secretion in inflammation
� Cytoprotective effects
� Four receptors: A1, A2A, A2B and A3
• Adenosine A3 Receptor (A3R)
� Gi protein-coupled receptor� Gi protein-coupled receptor
� cAMP
� Expressed in:
� Cells (PMNs, T cells, epithelial cells etc.)
� Tissue (Lung, Liver, Kidney etc.)
• A3R plays a pivotal role in cardiac, skeletal muscle, intestinal and kidney
ischemia-reperfusion injury
Hypothesis
• A3R activation attenuates lung IR injury
�Effects occur in part via a neutrophil-dependent
mechanismmechanism
• Pulmonary function
� Airway Resistance
Methods
In vivo mouse left lung hilar clamp model:
• 1 hr ischemia followed by 2 hrs reperfusion (IR)
• WT and A3R-/- mice: Sham
IR
IR+Cl-IB-MECA (A3R agonist; 100 µg/kg i.v.)
• Cytokine expression – BAL fluid
� Airway Resistance
� Pulmonary Compliance
� Pulmonary Artery Pressure
• Myeloperoxidase (MPO) – BAL fluid
• Edema – Lung wet/dry weight
• Neutrophil infiltration – Immunohistochemistry
A3R agonist improves lung function after IR injury
0
1
2
3
4
5
6
7
8
Pulmonary Compliance
( µµ µµL/cm H
20)
#
*
WT
A3R-/-
Sham IR IR+MECA IR IR+MECA
* p<0.05 vs. Sham
# p<0.05 vs. IRSham IR IR+MECA IR IR+MECA
Sham IR IR+MECA IR IR+MECA0
2
4
6
8
10
12
14
Pulmonary Artery
Pressure
(cm H
20)
*
#
0
0.5
1
1.5
2
Airway Resistance
(cm H
20/ µµ µµL/sec)
Sham IR IR+MECA IR IR+MECA
*
#
A3R agonist attenuates lung inflammation after IR
0
1000
2000
3000
4000
IL-6 (pg/mL)
WT
A3R-/-
#
*
0
200
400
600
800
1000
MIP-1αα αα(pg/mL) *
#
* p<0.05 vs. Sham
# p<0.05 vs. IR
0
Sham IR IR+MECA IR IR+MECA
0
2000
4000
6000
8000
Sham IR IR+MECA IR IR+MECA
KC (pg/mL)
*
#
0
Sham IR IR+MECA IR IR+MECA
A3R agonist decreases lung edema after IR
5
6
7
Weight Ratio
#
*
WT
A3R-/-
0
1
2
3
4
Sham IR IR+MECA IR IR+MECA
Lung Wet/Dry Weight Ratio
* p<0.05 vs. Sham
# p<0.05 vs. IR
A3R agonist decreases neutrophil infiltration in vivo
* p<0.05 vs. Sham
# p<0.05 vs. IR0
5
10
15
20
25
30
35
MPO (ng/mL)
#
*
WT
A3R-/-
WT (Sham) WT (IR) WT (IR+MECA)
0
10
20
30
40
50
WT-Sham WT-IR WT-IR+MECA
Neutrophils/HPF
#
*
A3R agonist decreases neutrophil activation in vitro
40
50
/mL)
WT
A3R-/-
#
*
In vitro model: Hypoxia/Reoxygenation (3/1 hr)
* p<0.05 vs. Normoxia
# p<0.05 vs. HR
0
10
20
30
Normoxia HR HR+MECA HR HR+MECA
MPO (ng/ #
A3R agonist decreases neutrophil chemotaxis in vitro
2
3
ChemotaxisIndex
(fold change)
#
*
* p<0.05 vs. Media + Vehicle
# p<0.05 vs. 10% FBS + Vehicle
0
1
NeutrophilChemotaxis
(fold change)
Media +
Vehicle10% FBS +
Vehicle
10% FBS +
MECA
Conclusions
� A3R activation attenuates lung dysfunction and inflammation
after lung IR.
� The protective effects of A3R activation are due, at least in
part, to attenuation of neutrophil activation and chemotaxis.
� The use of A3R agonists may be a novel therapeutic strategy
to prevent lung IR injury after transplantation.
Acknowledgments
Kron / Laubach Lab Members
Vanessa Hajzus
Tony Herring
Sheila Hammond
Cynthia Dodson
Funding:
• NIH R01: HL092953
• NIH T32: HL007849-I0 11A
