A homo-dimer of annexin V protects against ischemia reperfusion injury in lung transplantation

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Z Guan, DM Hwang, TK Waddell, M Cypel, M Liu, S Keshavjee.

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

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

REPERFUSION INJURY 10-20 % incidence 5x increase

3 Primary Graft Dysfunction (PGD) - a significant challenge in lung transplant Mechanism: ISCHEMIA REPERFUSION INJURY % incidence 5x increase in early postop mortality Inferior long term survival

4 Cell death is an important contributing factor in ischemia reperfusion injury Early phase apoptosis Phosphatidylserine (PS) externalization Caspase activation Late phase apoptosis DNA fragmentation (TUNEL positive) Membrane breakdown (PI positive)

5 S e n s itiv ity % S e n s itiv ity % Cell death markers (M30 and HMGB-1) in ex vivo lung perfusion perfusate predicts PGD 3 M30 HMGB-1 a t 1 h c h a n g e fr o m 1 h to 4 h AUC= % - S p e c ific ity % AUC= % - S p e c ific ity % Hashimoto / Keshavjee J Heart Lung Transpl April 2015.

6 Higher cell death signals detected during EVLP are associated with worse post transplant survival *p=0.03 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 7

8 Role of phosphatidylserine (PS) in ischemia reperfusion Capillary Blood flow Impaired Microcirculation PS Endothelial cells Anoxia / Apoptosis

9 Shielding of PS may prevent tissue injury after reperfusion Diannexin = Di (two) - Annexin V 9

10 Study Model Syngeneic single lung transplant in rats Pulmonary flush solution Donor 12 h cold ischemic time Reperfusion 2 hours Recipient transplant

11 Study groups Diannexin Group (DN group, n =10) Control Group (C group, n =10) Pulmonary flush solution Donor 12 h cold ischemic time Reperfusion 2 hours Recipient

12 Study groups Diannexin Group (DN group, n =10) Control Group (C group, n =10) Pulmonary flush solution + Diannexin Donor 12 h cold ischemic time Reperfusion 2 hours Recipient transplant I.V. Diannexin Diannexin: 1000 μg/kg

13 Study groups Diannexin Group (DN group, n =10) Control Group (C group, n =10) Pulmonary flush solution + Saline Donor 12 h cold ischemic time Reperfusion 2 hours Recipient transplant I.V. Saline

14 m m H g m m H g Diannexin Improved Gas Exchange Pulmonary vein po 2 Pulmonary vein pco * ** C g r o u p n =10 each D N g r o u p *p<0.01, **p=.04 0 C g r o u p D N g r o u p

15 Improved peak airway pressure with diannexin treatment * * * n =10 each *p<0.05

16 Diannexin binding was demonstrated in the lung grafts Transplanted lung Native lung At 2h after reperfusion Green: Diannexin; Blue: DAPI = nucleus

17 MSB s c o re H&E Reduced alveolar fibrin deposit by diannexin L u n g in ju ry s c o re control diannexin * 1 0 C D N C D N C D N C D N A lv e o la r fib r in A lv e o la r h e m o r r h a g e * p=0.04 n =10 each V a s c u la r c o n g e s t io n L e u k o c y te in filtr a tio n

Reduced cell death with diannexin treatment c le a v e d m o le c u le (p e rc e n ta g e d e n s ity ) C e lls /H P F n g /m L TUNEL staining 1 2 0 1 0 0 p=0.

18 Reduced cell death with diannexin treatment c le a v e d m o le c u le (p e rc e n ta g e d e n s ity ) C e lls /H P F n g /m L TUNEL staining p= PARP cleavage in the graft 0 C g r o u p D N g r o u p n =10 each Plasma M30 (caspase cleaved cytokeratin 18) *p< *p= C g r o u p D N g r o u p n =6 each 0 C g r o u p D N g r o u p n =10 each

19 m R N A /A c tin ra tio m R N A /A c tin ra tio IL -6 (p g ) / to ta l p ro te in ( g ) Lower pro-inflammatory cytokine expression in the diannexin treated group mrna Protein C g r o u p IL-6 *p=0.01 D N g r o u p MIP-2 *p=0.03 C g r o u p D N g r o u p IL-6IL-6 *p<0.01 C g r o u p D N g r o u p M IP -2 (p g ) / to ta l p ro te in ( g ) MIP-2 p=0.84 C g r o u p D N g r o u p n =10 each

20 Apoptotic cell death is an important component of ischemia reperfusion injury Shielding of exposed PS with diannexin: o o o o Decreased apoptotic cell death Improved lung injury Conclusion 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 20

School of Medicine Ishidsu Shun Memorial Scholarship Mitsukoshi Health

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

MASSACHUSETTS INSTITUTE OF TECHNOLOGY

Harvard-MIT Division of Health Sciences and Technology HST.54J: Quantitative Physiology: Organ Transport Systems Instructors: Roger Mark and Jose Venegas MASSACHUSETTS INSTITUTE OF TECHNOLOGY Departments