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

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

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.

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)

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.

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.

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

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

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

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

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.

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

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

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

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

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

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. CLICK 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

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.

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

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.

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

Thank you very much for your attention.