1. A homo-dimer of annexin V protects against ischemia reperfusion injury in lung transplantation
K Hashimoto, H Kim, H Oishi, M Chen, I Iskender, J Sakamoto, A Ohsumi, Z Guan, DM Hwang, TK Waddell, M Cypel, M Liu, S Keshavjee. Latner Thoracic Surgery Research Laboratories Division of Thoracic Surgery, Toronto General Hospital University of Toronto
I’d like to thank the AATS for the privilege of presenting our work

2. Disclosure
Diannexin for use in this study was provided by Astellas Pharma Inc.

3. Mechanism: ISCHEMIA REPERFUSION INJURY
Primary Graft Dysfunction (PGD)
- a significant challenge in lung transplant
Mechanism: ISCHEMIA REPERFUSION INJURY
10-20 % incidence
5x increase in early post- op mortality
Inferior long term survival
Lung transplantation is effective therapy for patients with end-stage lung disease. A major challenge in lung transplantation however is Primary Graft Dysfunction. PGD is the clinical manifestation of ischemia reperfusion injury. The incidence of PGD is still approximately 20% and is associated with significant early mortality and late graft dysfunction.

4. Cell death is an important contributing factor in ischemia reperfusion injury
Phosphatidylserine (PS) externalization
Caspase activation
Early phase
apoptosis
Cell death occurs in the context of ischemia reperfusion injury. Apoptosis is a form of cell death that is regulated in a series of well defined steps. In the late phase, cell membranes break down. This is loss of cell integrity associated with a secondary inflammatory reaction in response to released cytosolic contents.
Late phase
apoptosis
DNA fragmentation
(TUNEL positive)
Membrane breakdown
(PI positive)

5. Cell death markers (M30 and HMGB-1) in ex vivo lung perfusion perfusate predicts PGD 3
In fact, we have studied selected key biomarkers related to the cell death pathway such as M30 and HMGB-1 during pre-transplant ex vivo lung perfusion in human lung transplantation and have shown them to be highly predictive of severe grade 3 PGD.
AUC=0.78
AUC=0.86
Hashimoto / Keshavjee J Heart Lung Transpl April 2015.

6. Higher cell death signals detected during EVLP are associated with worse post transplant survival
Furthermore, patients who received lungs with M30 levels at 1h of EVLP in the upper third values had significantly worse 5 year survival than those who had M30 levels in the lower two thirds. Thus, if we could develop therapies to prevent cell death or “turn off” the switch to the programmed cell death cascade, we could improve the outcomes of lung transplantation….
Hashimoto / Keshavjee J Heart Lung Transpl April 2015.

7. Hypothesis
Diannexin can ameliorate ischemia reperfusion induced acute lung injury in lung transplantation by blocking progression of apoptosis
We hypothesized that the drug diannexin fulfill this role and ameliorate ischemia reperfusion-induced acute lung injury by blocking progression of the apoptotic cell death pathway

8. Role of phosphatidylserine (PS) in ischemia reperfusion
Capillary
Impaired Microcirculation PS
Blood flow ⇒
Phosphatidylserine is a cell membrane protein that is externalized in the early phase of apoptosis. This is important in the context of ischemia reperfusion injury.
CLICK
Externalized PS on reperfused endothelial cells binds circulating leukocytes and platelets and impairs the microcirculation. This induces further damage to the surrounding tissue.
Endothelial cells
Anoxia / Apoptosis

9. Shielding of PS may prevent tissue injury after reperfusion
Diannexin is an agent designed to combine two annexin V molecules. This dimer has a strong binding affinity to phosphatidylserine and prolonged half life in the circulation. This binding theoretically shields the exposed PS and prevents the recruitment of inflammatory cells thus ameliorating the subsequent tissue injury.
Diannexin = Di (two) - Annexin V 9

10. Study Model Syngeneic single lung transplant in rats
Pulmonary flush solution
12 h cold ischemic time
Donor
Reperfusion
Our hypothesis was tested in a syngeneic rat single lung transplant model, which is a standard model of ischemia reperfusion injury for lung transplantation. After the donor lung was flushed, the lungs were preserved in cold storage for 12h. The left lung was transplanted and reperfused for 2 hours.
2 hours
Recipient
transplant

11. Study groups Diannexin Group Control Group Pulmonary flush solution
(DN group, n =10)
Control Group
(C group, n =10)
12 h cold ischemic time
Recipient
Donor
Pulmonary flush solution
Reperfusion
2 hours
Two study groups were treated in a blinded fashion.

12. Study groups Diannexin Group Control Group Pulmonary flush solution
(DN group, n =10)
Control Group
(C group, n =10)
Pulmonary flush solution
+ Diannexin
12 h cold ischemic time
Donor
Reperfusion
In the study group, diannexin was added to the donor lung LPD flush solution, and also administrated intravenously to the recipient 5 min after reperfusion.
2 hours
Recipient
transplant
I.V. Diannexin
Diannexin: 1000 μg/kg

13. Study groups Diannexin Group Control Group Pulmonary flush solution
(DN group, n =10)
Control Group
(C group, n =10)
Pulmonary flush solution
+ Saline
12 h cold ischemic time
Donor
Reperfusion
In the control group, normal saline, was administered to the pulmonary flush solution and also at the time of reperfusion.
2 hours
Recipient
transplant
I.V. Saline

14. Diannexin Improved Gas Exchange
Pulmonary vein pO2
Pulmonary vein pCO2 * **
Here are our results. Lung graft oxygenation was significantly improved in the diannexin group as was the ventilation reflected in the carbon-dioxide level.
n =10 each
*p<0.01, **p=.04

15. Improved peak airway pressure with diannexin treatment* * *
Peak airway pressure in the graft was also significantly decreased in the diannexin group.
n =10 each
*p<0.05

16. Diannexin binding was demonstrated
in the lung grafts
Transplanted lung
Native lung
We immunostained for diannexin and indeed confirmed positivity in the systemically treated graft which is shown as green color in the left image. Much less diannexin was found in the native lung. These findings confirms there is PS exposure on the reperfused endothelial cells with the expected diannexin binding.
At 2h after reperfusion Green: Diannexin; Blue: DAPI = nucleus

17. Reduced alveolar fibrin deposit by diannexin
control
diannexin
H&E
MSB *
We assessed the degree of lung injury using a semi-quantitative histologic assessment. The alveolar fibrin score was significantly lower in the DN group.
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Using MSB fibrin staining, the alveolar deposits seen here in the control example are stained pink, confirming the presence of significantly more fibrin.
* p=0.04
n =10 each

18. Reduced cell death with diannexin treatment
TUNEL staining
p=0.15
n =10 each
*p=0.01
*p<0.01
PARP cleavage in the graft
Plasma M30 (caspase cleaved cytokeratin 18)
n =10 each
n =6 each
We interrogated various levels of the apoptosis pathway.
TUNEL, a marker of DNA fragmentation, tended to be lower in the DN group.
CLICK
The level of caspase cleaved PARP was significantly decreased in the DN group. Caspase-cleaved cytokeratin 18 in the plasma, which is the marker we have noted in our patients, was also significantly decreased in the diannexin group.

19. Lower pro-inflammatory cytokine expression in the diannexin treated group
mRNA
Protein
IL-6
MIP-2
IL-6
IL-6
MIP-2
*p=0.01
*p=0.03
*p<0.01
p=0.84
We assessed key pro-inflammatory cytokines in the graft that are known to play a role in the development of PGD. At the mRNA level, IL-6 and the il-8 analogue MIP-2, were significantly decreased in the diannexin-treated group. At the protein level, IL-6 was similarly significantly decreased in the diannexin group. MIP-2 was not significantly decreased and this discrepancy may be due to post - transcriptional factors.
n =10 each

20. Conclusion
Apoptotic cell death is an important component of ischemia reperfusion injury
Shielding of exposed PS with diannexin:
Decreased apoptotic cell death
Improved lung injury
Reduced the secondary inflammatory cytokine response
Significantly improved post transplant lung function
A promising potential therapy to be applied prior to or at the time of implantation to prevent and treat primary graft dysfunction
In summary, apoptotic cell death is an important component of ischemia reperfusion injury in lung transplantation.
We have shown that shielding the phosphotidyl serine that is exposed early in activation of the apoptotic pathway is beneficial in that: it decreased cell death, improved lung injury scores, decreased the inflammatory cytokine response and significantly improved transplanted graft function.
This appears to be a promising potential therapy to treat the donor lung and the recipient to prevent primary graft dysfunction after lung transplantation.

21. Acknowledgements Supervisor: Dr. S Keshavjee
Co-PI: Dr. M Liu, Dr. M Cypel
Latner Thoracic Surgery Research Laboratories
Keio University School of Medicine
Ishidsu Shun Memorial Scholarship
Mitsukoshi Health and Welfare Foundation
Obradovich Family Thoracic Surgery Research Scholarship
Thank you very much for your attention

22. Thank you very much for your attention.