Organ Repair and Regeneration: Preserving Organs in the Regenerative Medicine Era [1 ed.] 9780128194515, 0128194515

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ORGAN REPAIR AND REGENERATION PRESERVING ORGANS IN THE REGENERATIVE MEDICINE ERA

Edited by

Giuseppe Orlando, MD, PHD Marie Curie Fellow Department of Surgery Section of Transplantation Wake Forest Institute for Regenerative Medicine Wake Forest University School of Medicine Winston-Salem, NC, United States

Shaf Keshavjee, MD, MSc, FRCSC, FACS Surgeon-in-Chief, Sprott Surgery, UHN James Wallace McCutcheon Chair in Surgery Director, Toronto Lung Transplant Program Director, Latner Thoracic Research Laboratories Professor, Division of Thoracic Surgery and Institute of Biomedical Engineering Vice Chair Innovation, Department of Surgery University of Toronto Toronto, ON, Canada

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2021 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-819451-5 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Stacy Masucci Acquisitions Editor: Elizabeth Brown Editorial Project Manager: Pat Gonzalez Production Project Manager: Sreejith Viswanathan Cover designer: Matthew Limbert Typeset by TNQ Technologies

Contributors Masato Fujiyoshi Department of Hepato-PancreatoBiliary Surgery and Liver Transplantation, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

Stephen Large Royal Papworth Hospital, Cambridge, United Kingdom Simon Messer Royal Papworth Hospital, Cambridge, United Kingdom

Fanourios Georgiades Department of Surgery, Addenbrooke’s Hospital, University of Cambridge, Cambridge, United Kingdom

Aylin Acun Center for Engineering in Medicine, Harvard Medical School, Boston, MA, United States; Shriners Hospital for Children, Boston, MA, United States

Paula A. Grisales Wake Forest University School of Medicine, Winston Salem, NC, United States

Amish Asthana Wake Forest University School of Medicine, Winston Salem, NC, United States

Ahmer Hameed National Pancreas and Islet Transplant Laboratories, The Westmead Institute for Medical Research, Westmead, NSW, Australia; Department of Surgery, Westmead Clinical School, University of Sydney, Westmead Hospital, Westmead, NSW, Australia

Justine M. Aziz Wake Forest University School of Medicine, Winston Salem, NC, United States Lauren Brasile CSO, Research and Development, BREONICS, Inc., Albany, NY, United States; Bioscience, College of Nanoscale Science and Engineering, Albany, NY, United States

Wayne J. Hawthorne National Pancreas and Islet Transplant Laboratories, The Westmead Institute for Medical Research, Westmead, NSW, Australia; Department of Surgery, Westmead Clinical School, University of Sydney, Westmead Hospital, Westmead, NSW, Australia

Isabel M.A. Br€ uggenwirth Department of Surgery, Section of Hepato-Pancreato-Biliary Surgery and Liver Transplantation, University Medical Center Groningen, Groningen, The Netherlands Benedetta Bussolati Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy

Sarah A. Hosgood Department of Surgery, Addenbrooke’s Hospital, University of Cambridge, Cambridge, United Kingdom

Mark Clancy Transplant Unit, NHS Greater Glasgow & Clyde, Glasgow, United Kingdom; University of Glasgow, Glasgow, United Kingdom

Shaf Keshavjee Sprott Surgery, UHN, James Wallace McCutcheon Chair in Surgery; Toronto Lung Transplant Program; Latner Thoracic Research Laboratories; Division of Thoracic Surgery and Institute of Biomedical Engineering; Department of Surgery, University of Toronto, Toronto, ON, Canada

Vincent E. de Meijer Department of Hepato-Pancreato-Biliary Surgery and Liver Transplantation, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

Catherine La Pointe Sherbrooke University, Sherbrooke, QC, Canada

K. Dhital Heart Transplant Unit, St Vincent’s Hospital, Sydney, NSW, Australia; Transplantation Research Laboratory, Victor Chang Cardiac Research Institute, Sydney, NSW, Australia; Department of Medicine, University of New South Wales, Sydney, NSW, Australia

Jennifer Li Centre for Transplant and Renal Research, Westmead Institute for Medical Research, University of Sydney, Sydney, NSW, Australia

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Contributors

P.S. Macdonald Heart Transplant Unit, St Vincent’s Hospital, Sydney, NSW, Australia; Transplantation Research Laboratory, Victor Chang Cardiac Research Institute, Sydney, NSW, Australia; Department of Medicine, University of New South Wales, Sydney, NSW, Australia Domenica I. Marino Ohio State College of Arts and Science, Columbus, OH, United States Paulo N. Martins Department of Surgery, Division of Organ Transplantation, UMass Memorial Medical Center, University of Massachusetts, Worcester, MA, United States Laura Ioana Mazilescu Multi Organ Transplant Program, Department of Surgery, Toronto General Hospital, University Health Network, Toronto, ON, Canada; Division of Nephrology, The Hospital for Sick Children, Toronto, ON, Canada Sean M. Muir Wake Forest University School of Medicine, Winston Salem, NC, United States Michael L. Nicholson Department of Surgery, Addenbrooke’s Hospital, University of Cambridge, Cambridge, United Kingdom Giuseppe Orlando Department of Surgery, Section of Transplantation, Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, United States Paolo Porporato Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy Robert J. Porte Department of Hepato-PancreatoBiliary Surgery and Liver Transplantation, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

Siavash Raigani Division of Transplant Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States; Center for Engineering in Medicine, Harvard Medical School, Boston, MA, United States Natasha M. Rogers Centre for Transplant and Renal Research, Westmead Institute for Medical Research, University of Sydney, Sydney, NSW, Australia Andrea Rossi Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy S.E. Scheuer Heart Transplant Unit, St Vincent’s Hospital, Sydney, NSW, Australia; Transplantation Research Laboratory, Victor Chang Cardiac Research Institute, Sydney, NSW, Australia; Department of Medicine, University of New South Wales, Sydney, NSW, Australia Markus Selzner Multi Organ Transplant Program, Department of Surgery, Toronto General Hospital, University Health Network, Toronto, ON, Canada Basak Uygun Center for Engineering in Medicine, Harvard Medical School, Boston, MA, United States; Shriners Hospital for Children, Boston, MA, United States Korkut Uygun Center for Engineering in Medicine, Harvard Medical School, Boston, MA, United States; Shriners Hospital for Children, Boston, MA, United States Heidi Yeh Division of Transplant Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States Oscar Zou University of Sydney, Camperdown, Australia

Foreword

art of organ preservation in the regenerative medicine era of the transplant history (see the last chapter How the transplant landscape is changing in the regenerative medicine era), culminating in the vision and implementation of the most groundbreaking idea of the field, the organ repair center (see the chapter Implementing the Vision: the Organ Repair Center). We hope that the reader will enjoy this book and will eventually share our conviction that no fields in medicine has more interest in fostering progress in regenerative medicine more than organ transplantation, because the future of no other fielddmore than the future of transplantationdwill be forged by progress occurring in regenerative medicine. Regenerative medicine will change the way we think and do transplants, well beyond the instances of organ preservation and the organ repair center. This will take time, efforts, commitments, investments, and partnership with the major regenerative medicine societies. However, as of now, organ preservation offers a formidable opportunity for collaboration to the two fields.

Regenerative and transplant medicine share the same parenthood (Alexis Carrell is acknowledged as the father of both regenerative and transplant medicine), speak the same language, and pursue the same objectives. If we accept the most popular definition of the term regenerative medicine, according to which it is the field of health sciences that “aims at repairing or replacing damaged cells, tissues, or organs,” we can understand how intimately interconnected the two fields are. Ante literam, transplant medicine has pursued one of the major regenerative medicine objectives (to replace damage organs, tissues, or cells) for more than a century, since its very early days. However, if we pay more attention on the history of transplantation and the semantic of the word “repair,” we realize that we have also pursued the other major objective mentioned above, namely the “repair” of damaged cells, tissues, or organs. In fact, the cells, tissues, and organs that are procured for transplant purposes are subjected to a massive stress during the death of the donor, the procurement surgery, and the preservation and the implantation in the recipient. In order to minimize the deriving damage, we have developed a surrogate field of science called “organ preservation” thatdto the editors of this bookd represents the most formidable platform nowadays for the application of regenerative medicine technologies in transplant medicine. This book explains this concept and draws the state of the

Anthony Atala, MD Wake Forest School of Medicine G. Link Professor and Director Wake Forest Institute for Regenerative Medicine W.H. Boyce Professor and Chair Department of Urology

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Preface

Regenerative and transplant medicine share the same parenthood (Alexis Carrell is acknowledged as the father of both regenerative and transplant medicine), speak the same language, and pursue the same objectives. If we accept the most popular definition of the term regenerative medicine, according to which it is the field of health sciences that “aims at repairing or replacing damaged cells, tissues, or organs,” we can understand how intimately interconnected the two fields are. Ante literam, transplant medicine, has pursued one of the major regenerative medicine objectives (to replace damaged organs, tissues, or cells) for more than a century since its very early days. However, if we pay more attention to the history of transplantation and the semantic of the word “repair,” we realize that we have also pursued the other major objective mentioned earlier, namely the “repair” of damaged cells, tissues, or organs. In fact, the cells, tissues, and organs that are procured for transplant purposes are subjected to massive stress during the death of the donor, the procurement surgery, the preservation, and the implantation in the recipient. To minimize the deriving damage, we have developed a surrogate field of science called “organ preservation” thatdto the editors of this bookdrepresents the most formidable platform nowadays for the application of regenerative medicine technologies in transplant medicine. This book explains this concept and draws the state of the art of organ preservation in the regenerative medicine era of the transplant history (see the last chapter How the transplant landscape is changing in the regenerative medicine era), culminating in the vision and implementation of the most groundbreaking idea of the field,

the organ repair center (see the chapter Implementing the Vision: the Organ Repair Center). We hope that the reader will enjoy the book and will eventually share our conviction that no fields in medicine have more interest in fostering progress in regenerative medicine more than organ transplantation because the future of no other fielddmore than the future of transplantationdwill be forged by progress occurring in regenerative medicine. Regenerative medicine will change the way we think and do transplants, well beyond the instances of organ preservation and the organ repair center. This will take time, efforts, commitments, investments, and partnerships with the major regenerative medicine societies. However, as of now, organ preservation offers a formidable opportunity for collaboration in the two fields.

xiii

Giuseppe Orlando, MD, PHD Marie Curie Fellow Department of Surgery Section of Transplantation Wake Forest Institute for Regenerative Medicine Wake Forest University School of Medicine Winston-Salem, NC, United States Shaf Keshavjee, MD, MSc, FRCSC, FACS Surgeon-in-Chief, Sprott Surgery UHN James Wallace McCutcheon Chair in Surgery Director, Toronto Lung Transplant Program Director, Latner Thoracic Research Laboratories Professor Division of Thoracic Surgery and Institute of Biomedical Engineering Vice Chair Innovation, Department of Surgery University of Toronto Toronto, ON, Canada

C H A P T E R

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Ischemia-reperfusion injury Jennifer Li1, Natasha M. Rogers1, Wayne J. Hawthorne2,3 1

Centre for Transplant and Renal Research, Westmead Institute for Medical Research, University of Sydney, Sydney, NSW, Australia; 2National Pancreas and Islet Transplant Laboratories, The Westmead Institute for Medical Research, Westmead, NSW, Australia; 3Department of Surgery, Westmead Clinical School, University of Sydney, Westmead Hospital, Westmead, NSW, Australia

Concepts and molecular mechanisms of ischemia reperfusion injury

cell stunning or hibernation)dand irreversible damage or necrosis if the cell’s adaptive threshold to utilize anaerobic metabolism is overwhelmed. The following reperfusion phase, on one hand, overcomes this ischemic insult by restoring blood flow to salvage viable ischemic tissue but this can further exacerbate injury through the generation of reactive oxygen species and an intensified immune response and inflammation (Fig. 1.1) [1]. This concept and the underlying molecular mechanisms are applicable to all the major organs, although there are appreciable organ-specific differences in the sensitivity, severity, and reversibility of IRI. It is no surprise, given the metabolic activity of the brain and heart, that these are the most susceptible IRI and clinically this is commonly seen as ischemia cerebral vascular accidents (stroke) and myocardial infarction. Following an episode of myocardial infarct, there will be a portion of irreversible cardiomyocyte damage, but also a portion of viable tissue that displays the phenomenon of stunningda phenotype characterized by a persistent period of dysfunction postreperfusion [2]. These viable cells then can enter a hibernation phase following prolonged

Ischemia-reperfusion injury (IRI) is an important concept and is an important mechanism that leads to cell dysfunction, cell death, tissue injury, and organ dysfunction on the basis of the disrupted blood flow profile and the inability to deliver the required oxygen and nutrient contents to the affected cells. IRI has important implications for clinical medicine and this chapter will outline the fundamental concepts of the underlying cellular and molecular mechanisms of cell death, danger signals, cellular inflammation, and reactive oxygen species. This will then be followed by a discussion of IRI applied in the clinical setting of nephrology, as the prototypical example to highlight research and developments on this topic.

Fundamental concept of ischemiareperfusion injury The fundamental concept is that damage in IRI occurs in a bimodal fashion. The initial ischemic phase leads to cellular dysfunction (including

Organ Repair and Regeneration https://doi.org/10.1016/B978-0-12-819451-5.00012-3

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Copyright © 2021 Elsevier Inc. All rights reserved.

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1. Ischemia-reperfusion injury

FIGURE 1.1 The key concept: biphasic ischemia-reperfusion injury. There are ischemic damage and necrosis during the ischemic phase and also further injury during the reperfusion phase due to the generation of reactive oxygen species and inflammation. The sensitivity or susceptibility of organs to IRI is variable and usually highest in the most metabolically active organs. Created with BioRender.

ischemia, characterized by an adaptive metabolic phenotype for utilization of glycolysis or anaerobic metabolism as their adenosine 50 -triphosphate (ATP) energy source [3,4]. The kidneys are also an organ of high metabolic demand and the proximal tubular cells are the most sensitive to changes in oxygenation (as opposed to medullary cells that can tolerate ischemia and glycolysis). In comparison, hepatocytes are relatively resistant to IRI, possibly due to the servient high carbohydrate stores in the liver. The IRI risk of the intestinal mucosa varies with location and blood supply, with the highest perfusion gradient and sensitivity to IRI in the proximal segments of bowel supplied by the superior mesenteric artery [5,6].

Causes of ischemia-reperfusion injury As mentioned, the ischemic phase of IRI occurs due to the cessation of blood flow to the affected organ or tissue. This leads to a mismatch between oxygen and nutrient delivery, whereas cellular demands lead to ineffective aerobic metabolism

and reliance on anaerobic metabolism and glycolysisdwhich generates insufficient energyrich phosphates (such as ATP and phosphocreatine) to support many essential cellular processes. There is also disruption to the maintenance of cellular osmolality, membrane electrochemical polarity, acid/base control, and homeostasis of activation/inactivation pathways, which are all dependent on energy-dependent ion pumps found either on the cell surface membrane or intracellular organellesdthe effects cumulating to cell death. Although necrosis is the major form of cell death in IRI, pyroptosis, necroptosis, apoptosis, and autophagy all have an important role in the pathophysiology of IRI. The switch to glycolysis also leads to the accumulation of waste products, including lactate (leading to the acidotic environment) and hypoxanthine (substrate for reactive oxygen species formation), which further compound the insult of ischemia. Many clinical scenarios lead to cessation of blood flow or severe hypoperfusion to set up

Concepts and molecular mechanisms of ischemia reperfusion injury

the hypoxic and nutrient deplete environment of IRI and they can be categorized mechanistically: 1. Intrinsic occlusion of the blood vessel: cerebral vascular accidents (“ischemic stroke”) with occlusion of a major cerebral vessel due to either embolic or atherosclerotic disease; acute myocardial infarction with occlusion of the coronary artery from ruptured plaque and underlying heavy atherosclerotic disease; mesenteric ischemia from occlusion of blood supply to the intestines; or critical limb ischemia in the setting of severe peripheral vascular disease, often in smokers and poorly controlled diabetic disease. 2. Extrinsic occlusion of the blood vessel: mass effect or compartment syndrome causing occlusion of the blood vessel due to expansion of the adjacent tissue, usually from edema or a hematoma (crush injury, major trauma, and vascular surgery). 3. Globally reduced cardiac output or blood pressure: shock states (such as cardiogenic shock from cardiac arrest; septic shock from severe, uncontrolled infection; hypovolemic shock from massive blood loss) can lead to reduced cardiac output and blood flow to all organs in the body. Another example would be cardiothoracic bypass surgery, with deliberate aortic cross-clamping that results in systemic ischemia. 4. Transplantation: During organ procurement in transplantation, donor organs are either preserved in cold storage or machine perfusion techniques (hypothermic or normothermic) for transportation and preparation for recipient donor surgery. Cold ischemic time refers to the time spent in cold storage (ice) from organ retrieval to reanastomosis to the recipient to restore blood flowdand this is a major contributor to delayed graft function. Warm ischemic time refers to any period of reduced or inadequate perfusion of the organ with “warm” blood and can be split into donor related (reduced perfusion due to donation after cardiac death)

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and surgery/donor related (during reanastomosis with the venous supply connected before the arterial supply, or hypotensive states due to blood loss in the recipient perioperatively). There is the restoration of blood flow and replenishment of oxygen and nutrient substrates in the reperfusion phase. Although this halts further ischemic damage to viable cells, it paradoxically aggravates additional cellular and tissue injury through the generation of reactive oxygen species and cellular inflammation, the concepts that will be explained in more detail in the following section.

Reactive oxygen species in ischemiareperfusion injury Reactive oxygen species (ROS) are critical in the pathophysiology of IRI. The primary ROS moieties released in IRI are superoxide and hydrogen peroxide (enzymatically dismutated superoxide), which interact with various lipids and proteins to cause oxidative stress [7]. Superoxide will also interact with bioavailable nitric oxide (NO) to form reactive nitrogen species (RNS) such as peroxynitrite. ROS and RNS contribute to cellular dysfunction, impaired vascular tone, and tissue damage and can also act as dangerassociated molecular pathogen (DAMP) signals to further promote the inflammatory cascade [8]. In addition to peroxynitrite, other important biologically active ROS moieties include malondialdehyde, conjugated dienes, hydroxynonenal, and oxidized glutathione. Inducible nitric oxide synthase (iNOS) is activated by inflammation and the endothelial (eNOS) isoform is important for the regulation of vascular muscle tone and generation of superoxide when uncoupled in the absence of essential cofactors. iNOS is found in all inflammatory cells and produces large amounts of NO to generate peroxynitrite [9,10]. Major sources of superoxide include NADPH oxidase (NOXdexpressed in phagocytic cells such

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1. Ischemia-reperfusion injury

as macrophages and neutrophils) and mitochondrial cytochrome P450 peroxidases (mcP450). NOX knockout mice suffer less injury following IRI in the kidneys [11,12], but also this effect is seen in the myocardium [13], lung [14], and liver [15]. Superoxide is readily converted into hydrogen peroxide by superoxide dismutase (SOD-2), localized in the outer mitochondrial membrane [16,17], and mcP450 further metabolizes hydrogen peroxide into highly proinflammatory hypobromous and hypochlorous acid [18], uncoupled endothelial nitric oxide synthase, and xanthine oxidase. Furthermore, excess ROS in IRI can lead to the accumulation of dynamin 1 like protein (DRP1) in the mitochondrial membrane, further exacerbating

TABLE 1.1

mitochondrial fragmentation, the release of mitochondrial DNA resulting in both mitophagy and cell death (Table 1.1) [16,19,20]. Other important ROS pathways include xanthine oxidase (for purine metabolism) and heme oxygenase (degradation of heme to bilirubin to release iron and carbon monoxide [21]. Inhibition of xanthine oxidase [22] and expression of heme oxygenase in the renal parenchyma [23] or infiltrating myeloid cells [24] have all shown beneficial protection in the setting of renal IRI. Interventions that enhance ROS scavenging are universally protective against IRI [25e27]. However, no ROS-mediating agents have performed sufficiently effectively in clinical trials to reach clinical use [28e31].

Brief Summary of Source of Superoxide and Reactive Oxygen Species.

Sources

Location

Comments

NADPH oxidases (NOX)

Macrophages and neutrophils

Membrane-bound amalgamated subunits such as NOX 1e5, Duox 1 & 2. These generate superoxide and modulate damage by conversion of xanthine oxidase and uncoupling of endothelial nitric oxide synthase

Cytochrome P450 enzymes

Liver predominant

CYP450 enzymes use oxygen or NADPH to alter the redox status of lipids, steroids, and vitamins. They are also found as eosinophil peroxidase and neutrophil myeloperoxidase that produce hypobromous and hypochlorous acid

Mitochondrial oxidative phosphorylation

Mitochondria

Electron leak from complexes I and III from the mitochondrial electron transport chain causes the reduction of oxygen to superoxide Superoxide dismutase (SOD) and monoamide oxidases are also found in the mitochondrial membrane and produce hydrogen peroxide and p66shc that oxidizes cytochrome C

Nitric oxide synthase (NOS)

Neuronal NOS Inducible NOS Endothelial NOS

nNOS is constitutively expressed, iNOS is induced with inflammation and eNOS is critical in the regulation of vascular tone. These enzymes require the cofactor tetrahydrobiopterin to oxidize L-arginine to L-citrulline and produce nitric oxide (NO). Excess NO can combine with superoxide to generate peroxynitrite, which further mediates ROS damage

Xanthine oxidase (XO)

Variable, highest in the endothelium

Xanthine dehydrogenase (XDH) undergoes translation in the setting of inflammation into xanthine oxidase (XO) and can generate superoxide and hydrogen peroxide

Heme oxygenase (HO)

Variable

Heme oxygenase is usually undetectable at basal levels but upregulated in response to IRI and is critical for cytoprotectiondantioxidant, antiinflammatory, and antiapoptotic capacity

Concepts and molecular mechanisms of ischemia reperfusion injury

Cell death pathways in ischemiareperfusion injury Not all cell death pathways are made equald the mode of cell death influences and manner of release of intracellular contents in response to injury during IRI influences its proinflammatory potential and this is summarized in Fig. 1.2. Necrosis and necroptosis Necrosis is the major pathway of cell death in IRI, a form of unregulated cell death in response to excessive ischemia and depletion of ATP stores. It occurs through both (1) the generation of reactive oxygen species (ROS), which overwhelms the cell’s antioxidant capacity; and (2) activation of calcium-dependent proteases

FIGURE 1.2

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(including calpain) due to increased intracellular calcium levels from both reduced uptakes in the endoplasmic reticulum and disruption of the inner mitochondrial membrane. Both ROS and calcium-dependent proteases lead to damage and breakdown of lysosomal and plasma membranesdleading to cellular and organelle swelling and release intracellular contents to expose DAMPs, which drive a robust inflammatory response [1,32e35]. Although this is the traditional view of cell death in ischemia, there are cells that exhibit the typical plasma membrane destruction and release of DAMP signals like necrosis but morphologically retain an intact nucleus. This is termed necroptosis, which introduces the concept of necrosis with a component necrosis

Key cell death pathways in ischemia-reperfusion injury. Of these, necroptosis, necrosis, and pyroptosis have the highest proinflammatory potential and have received most research attention. Created with BioRender.

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1. Ischemia-reperfusion injury

that may have a component of regulated cell deathdakin to apoptosis but independent of caspase activity. Necroptosis can be initiated in IRI through ischemia, oxidative stress, cytokines such as tumor necrosis factor (TNF), and further mediated via immune ligands, including TNF proteins (TNF-⍺, Fas ligands), lipopolysaccharide (LPS), TNF superfamily receptors, toll-like receptors (particularly TLR3 and TLR4), and interferon receptors. Independent of caspase, in particular, if there is the absence of caspase-8, the multimerization of Fasassociated protein with death domain (FADD) will preferentially recruit receptor-interacting serine/threonine kinase (RIPK1 and RIPK3) to initiate necroptosis via the substratedmixed lineage kinase domain-like protein (MLKL) [36]. Furthermore, RIPK3 can activate the NLRP3 inflammasome complex (NLPR3: NACHT, LRR, and PYD domains-containing protein 3) to subsequently activate caspases (caspase-1 and caspase-11), which can initiate pyroptosis, which is discussed in the next section. These mechanisms are supported by the animal studies that demonstrated increased necroptosis following IRI and transplantation in caspase-8-deficient mice and that RIPK3 knockout mice exhibited less IRI, necrosis, fibrosis, and HMGB1 levels [37]. Necrostatin-1 (a RIPK1 kinase inhibitor) has also been shown to limit necroptosis in preclinical studies including for cerebral and myocardial IRI [38] but have had varying results in renal IRI [39,40]. Although there are currently phase 2 clinical trials underway for necrostatin (or GSK2982772) in ulcerative colitis [41], there is a limited therapeutic opportunity in the setting of IRI given the rapid progression of the necroptosome signaling cascadedbut necrostatin potentially could be used if IRI injury is anticipated, such as major cardiac surgery or solid organ transplantation. Pyroptosis Pyroptosis leads to lytic cell death similar to necrosis but fundamentally differs based on its dependence on caspase-1 [42]. It has been shown

to be crucial to control bacterial infections [43] and HIV-induced cell death [44] but may have an important role in IRI. Pyroptosis can lead to activation of inflammasomes (multimeric protein complexes), with a role in both parenchymal injury, recruitment of additional adaptor proteins that cleave caspase-1 to trigger further pyroptosis and clearance of cellular debris in the reparative phase. Other caspases have been shown in the pyroptosis, including caspase 11 (mice) through the noncanonical pathway and caspase-3, 4, and 5 (humans). Functionally, these caspases cleave and activate gasdermin to release the effector N-terminal domain from the inhibitory effects of its C-terminal domain. Of these, caspase-1, 4, 5, and 11 all target gasdermin-D and caspase-3 targets gasdermin-E [45] and facilitate the release of the N-terminal to bind to acidic phospholipids of the interior aspect of the plasma membrane and subsequent pore formation to release proinflammatory cytokines IL-1b and IL-18 [46]. Interestingly, caspase-3 is also important in the regulation of the apoptosis pathway [47], and these cell death pathways are unlikely to act independently of each other. This is demonstrated by a mouse kidney transplant model, as there was reduced caspase-3 and less histological injury when Q-VD-OPh (a pan-caspase inhibitor) was added to the cold perfusion fluid of the donor kidney graft [48] but the use of systemic zVAD, another pancaspase inhibitor, did not confer protection against IRIdraising the possibility of significant caspase-8 inhibition, thus promoting the activation of necroptosis pathway [39]. Apoptosis and autophagy Apoptosis, qualitatively distinct and less common than necrosis following IRI, generates a more immunologically tolerant environment. It is characterized by cell shrinkage, chromatin condensation, plasma membrane blebbing, and apoptotic bodies [49]. Exposure of phosphatidylserine on the cell surface functions as a “find me, eat me” signal to attract macrophage for

Concepts and molecular mechanisms of ischemia reperfusion injury

efferocytosis and clearance. The initiation of apoptosis can be via the intrinsic or extrinsic pathways in IRI. The extrinsic pathway involves activation of death factors of the TNF-family ligands (TNFa, TNF-related apoptosis-inducing ligand or TRAIL, and Fas-ligands or Fas-L) and cell surface receptors (TNF-R1, TRAIL-R1, TRAIL-R2, and Fas) [50,51] with subsequent activation of initiator and executioner caspases. This then leads to oligomerization of the cytoplasmic regions of death receptors including FADD, procaspase-8, and cellular FADD-like ICE (c-FLIP). The intrinsic pathways of apoptosis in response to mitochondrial stress lead to the damage of its outer membrane and release of cytochrome C, which combines with the apoptotic protease activating factor (Apaf-1) to initiate apoptosome complex formation, procaspase-9 recruitment, autolytic cleavage, and activation. Apoptosis is regulated primarily by B-cell lymphoma 2 (Bcl-2) family, which is either pro- or antiapoptotic based on the homology domains (BH) and can be divided into three main subsets: pro-apoptotic members (Bim, Bid Bad, and Puma), pro-apoptotic effector molecules (Bax and Bak), and antiapoptotic proteins (Bcl-2, Bcl-xl, Bcl-G) [52]. These subsets are also regulated by p53, an important tumor suppressor gene involved in apoptosis. Trials using QPI-1002 to silence p53 is currently in progress for both DGF following transplantation and AKI following major cardiac surgery. Finally, autophagy (or “self-eating”) is essential to prevent the formation of damaging, cytotoxic protein aggregates by degradation (and clearance) of misfolded or damaged cellular components [53]. It is initiated in response to external stimuli, such as the hypoxia and nutrient deprivation of IRI. Autophagy is typically initiated by exogenous stimuli such as hypoxia, nutrient deprivation, increased temperatures, or high energy demand. There are currently three recognized forms of autophagy: macroautophagy (commonly called as autophagy), microautophagy (engulfment of cytoplasmic contents directly

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by lysosomes via invagination), and chaperonemediated autophagy (where proteins are targeted by HSP70 and transported to the lysosome). The process of autophagy follows the following five steps, including (1) nucleation of the double membrane phagophore; (2) expansion of the phagophore to engulf intracellular components; (3) maturation of the phagophore into an autophagosome; (4) fusion of autophagosome with lysosome to form autolysosomes; and (5) lysosomal degradation of engulfed cytosolic elements with endproducts, such as amino acids and fatty acids recycled in de novo protein synthesis or energy production via Krebs cycle and gluconeogenesis [54]. Not surprisingly, this mechanism is rapidly induced in IRI to protect cells from injury and death [55,56] and is highly regulated by the autophagy-related genes (Atg) [57]. Pharmacological inhibition of autophagy (hydroxychloroquine or 3-methyladenine) has shown conflicting results, with reports of both exacerbation [58] and protection [59] from IRIdlikely due to the degree of inhibition and risk of accumulated damaged mitochondria and ubiquitin-positive protein aggregates [60]. Suppression of ATG5 by doxycycline [61], or induction by FGF-10 [62] or rapamycin (mTOR inhibitor) [63] was associated with worse outcomes postmouse renal IRI, which suggest possible ATG-specific roles. The effects of autophagy on other tissues are no clearer, as studies of this pathway in hepatic IRI [61,64e67] and myocardial IRI [68] also show differing results. Although autophagy is crucial for cardiac development (embryonic loss of Atg5, Atg7, or Beclin-1 leads to structural abnormalities) [69], preconditioning of cardiomyocytes with rapamycin (mTOR inhibitor) induces autophagy and confers protection from IRI [70] but downregulation of autophagy can prevent cellular death and promote cardiac repair [71]. Clearly, autophagy is important in IRI but further research is needed to clarify the specific roles and potential as therapeutic targets in the future.

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1. Ischemia-reperfusion injury

Danger signals: the link between cell death to inflammation DAMP molecules bind to pattern recognition receptors (PRRs) and is the basis of activation of the innate immune system in response to tissue and cellular injury. This is in the absence of microbes or pathogens (pathogen-associated molecular pattern (PAMPs)) and both DAMPs and PAMPs share or have closely related binding sites and downstream transduction pathways (Fig. 1.3). Danger-associated molecular patterns Under physiological conditions, intracellular DAMPs are normally sequestered within the cell and not visible to the immune system, but

these are released into the extracellular environment following disruption of the cell membrane following cellular stress, injury, or death (via pyroptosis, necrosis, or necroptosis and to a limited extent apoptosis in the setting of IRI). Extracellular DAMPS can be generated through proteolytic cleavage of matrix proteins and join a long list of DAMPs implicated to bind to PRR in invoking immune response in the sterile environment of IRI. Fibrinogen [72] and HMGB1 [73,74] are two examples of important DAMPs in IRI. High levels of DAMPs can be detected locally and systemically in humans and animal studies, and exogenous administration of DAMPs can exacerbate IRI injury, whereas inhibition of DAMPs can reduce the severity of injury [75].

FIGURE 1.3 Key damage-associated molecular patterns (DAMP) released following cell death in ischemia-reperfusion injury. These subsequently bind to various pattern recognition receptors (PRR) to further promote the inflammatory cascade. Created with BioRender.

Concepts and molecular mechanisms of ischemia reperfusion injury

Pattern recognition receptors DAMPs bind to PRR, as the initial step of the signaling pathway, but the specificity and sensitivity of various PRRs to individual DAMPs are not well studied. From the available research, the two most important PRRs for IRI include cytoplasmic NLR (nucleotide oligomerization domain (NOD)-like receptors) and transmembrane toll-like receptors (TLR). Other PRRs important more so for antiviral and antimicrobial protection include RLR (retinoic acid-inducible gene (RIG)-like receptors) and ALR (absent in melanoma 2 (AIM2)-like receptors) [76]. C-type lectin receptors (CLR), typically work against invading pathogens but also have a role in IRI, as they can synergize with TLR to amplify the cytokine production and sense necrosis via Ctype lectin 4e [77] and are also expressed on dendritic cells as CL2C9a or MINCLe [78]. DAMP and PRR interactiondcell signaling TLR is the most important PRR in IRI, as both TLR2 and TLR4 induce cytokine production in response to intracellular targets released following cell death [79]. There likely is tissue or organ-specific effects of TLR, as global knockout of TLR2 and TLR4 is protective against renal [80,81] and myocardial [82] IRI but was associated with decreased survival with lung IRI [79]. In addition to TLR binding, DAMPs often require varying coreceptor and adaptor molecules, such as CD14, MD-2, and NLRP3, for effective downstream signal transduction [75]. Subsequent downstream signal transduction involves five important adaptor molecules, including myeloid differentiation factor 88 (MyD88), MyD88-adaptor like (Mal), TIR domain-containing adaptor inducing IFN-beta (TRIF), TRIF-related adaptor molecule (TRAM), and sterile alpha and HEAT-armadillo motifs (SARM) [83]. The MyD88-dependent pathway is activated by all TLR molecules (except TLR3) and requires IL-1R-associated kinases (IRAK-1, -4), TNF receptorassociated factor 6 (TRAF-6), and mitogen-activated

9

kinases (MAPK) to eventually activate the NFkB transcription factor to drive inflammation. There also exists a MyD88-independent pathway (via TRIF) that can be activated via both TLR3 or TLR4 and the interferon regulatory factor (IRF) family of transcription factors. DAMP and PRR interactiondinflammasome formation Binding of DAMP signals to intracellular or cytosolic receptors such as NLR or ALR is the initial step of inflammasome formation. The NLRP inflammasome is a complex consisting of (1) the NLR receptor; (2) the adaptor proteind apoptosis-associated speck-like protein containing caspase recruitment domain (ASC); and (3) the effector protein (typically procaspase-1). Of the inflammasomes, NLRP3 is the bestcharacterized complex in IRI and critical in the control of pyroptosis. It is expressed in both renal tubular epithelial cells and the infiltrating leukocytes and the NLRP3 assembly can proteolytically cleave enzymes, such as procaspase-1, prointerleukin-1b and 18 into the active form, which acts to recruit immune cells, particularly of the innate system to the site of injury. Studies in caspase-1, NLRP3, ASC, and IL-18 deficient mice demonstrated protection against renal injury [84e88] but the use of pharmacological inhibitors of involved processes have shown mixed resultsdhydroxychloroquine (downregulates cathepsin leading to suppression of NLRP3) [59] showed protection, while anakinra (antibody blockade of the downstream IL-1 receptor) did not achieve significant effects [84]. The role of NLRP3 inflammasome in the liver [89] and myocardial [90,91] IRI is still not yet clear from the currently available research.

Cellular inflammation in ischemiareperfusion injury The other key player in IRI is cellular infiltration and inflammation. The microvasculature is

10

1. Ischemia-reperfusion injury

a key component affected in IRI and although there are organ-specific structural differences, ultimately, endothelial damage and increased vascular permeability (through alterations of the glycocalyx and cytoskeletal elements of cell-cell interactions), upregulation of P-selectin (initiates cell rolling), ICAM-1 (cell adhesion), and PECAM-1 (diapedesis) to facilitate transmigration of leukocytes [92], as summarized in Fig. 1.4. Innate immune response The inflammatory cell population has temporal variation during the course of IRI, but both neutrophils and natural killer T (NKT) cells are the first primary responders in IRI, followed by monocyte recruitment.

DAMP signals such as high mobility group box 1 (HMGB1) can induce neutrophil NETosis, or release of its chromatin granular contents (including proteinases and cationic peptides), reactive oxygen species, and chemokines and cytokines to exacerbate and perpetuate tissue injury. The role of neutrophls in IRI is yet to be definitely delieated, as studies have shown neutropenia to be protective in cardiac [93], hepatic [94], pulmonary [95] and intestinal [96] IRI. The targeting of neutrophil recruitment molecules (such as CD44 [97], ICAM-1 [98] and cathepsin G [99]) also confer a degree of renal protection e but neutrophil-depleted animals were still susceptible to IRI. [100,101] While there is no doubt that neutrophils are important in early phases of

FIGURE 1.4 Summary of inflammation in ischemia-reperfusion injury. Severe ischemia results in cellular necrosis and release of DAMP signals, self-peptides, and reactive oxygen species. This is followed by cellular adhesion, migration, and transmigration from the microcirculation to the site of injury. These innate cells further promote the inflammatory cascade through the release of various chemokines and cytokines. Dendritic cells are the most important antigen-presenting cells (either derived from tissue resident or circulating populations) that link the innate and adaptive immune system. Created with BioRender.

Concepts and molecular mechanisms of ischemia reperfusion injury

inflammation and tissue damage, the lack of protection in the absence of neutrophils suggests their role is not solely limited to this and have a role in orchestrating downstream events. NKT cells are a unique subset of the T-cell populationdthey express both CD161 (NK1.1 as the murine homolog) and a T-cell receptor but do not recognize peptides associated with antigen-presenting cell and major histocompatibility complex (MHC) molecules. They do, however, respond to glycolipid presented by CD1d [102,103] and produce substantial proinflammatory Th-1 type (IFN-g, TNF-a) and Th2 (IL-4, IL13) cytokines and can regulate both dendritic and T-cell functions [104]. Inhibition of NKT cells has shown benefit in protection from hepatic IRI [105] but the role in other organ IRI is yet to be defined. Natural killer (NK) cells are also seen early in the inflammatory process and have the direct cytotoxic capacity [106] but their role in IRI is uncertain. CD137þ NK cells can stimulate renal tubular epithelial cells to express CD137-ligand and CXCR2 to induce neutrophil migration (Kim 2012), and tubular epithelial cells themselves can produce CCR5 that is required for NK cell chemotaxis (Kim, 2013) but further research is required to elucidate their role. Monocyte recruitment is rapidly followed by differentiation in the macrophages, including the M1 macrophage phenotype that is activated by various stimuli, including PRR and DAMP signaling, reactive oxygen species, chemokines, and IFN-g released by T-helper and NKT cells [107], as opposed to the alternate M2 macrophage phenotype, which has wound healing and immunoregulatory capacity. Finally, dendritic cells (DC) also have a critical role in both injury and reparative phases of IRI and can be derived from the circulating (bone marrow derived and seen in the first 3 h post IRI) or tissue derived (or tissue-resident) pool of dendritic cells. They are important sentinels and are the most effective antigen-presenting cells to provide a link between the innate and adaptive immune response. DCs respond to

11

both DAMP signals (such as HMGB and HSP via TLRs) and antigens (self-antigen in native IRI, or foreign peptides in the setting of IRI or alloimmune response in solid organ transplantation), which are then processed to form the MHC complexes to present to the T-cell receptor. These stimulated DCs have an increased costimulatory capacity through maturation and upregulation of costimulatory molecules (CD40, CD80, and CD86) and can also promote local inflammation by the production of NF-kB-related cytokines following the activation of the TLR-MyD88 pathways). Previous studies have shown depletion of DCs in transgenic mice (via CD11cdiphtheria toxin) demonstrating less biochemical or histological injury following renal IRI [108]. Dendritic cells can also display an immunomodulatory phenotype and may be important for the reparative phase via the production of antiinflammatory cytokines (IL-10) and induction of regulatory T-cells [109,110]. DCs can also activate NK-T cells, and this seems to be regulated by sphingosine-1-phosphate (S1P), a ligand of G-coupled protein receptors and can influence neutrophil recruitment and proinflammatory cytokine production, such as IFNgamma in IRI [111]. S1P-receptor three knockout mice were protected from renal IRI, and this was postulated to be achieved through several mechanismsdincluding the reduced release of IFN-gamma and IL-6 from intracellular stores; and increase of both IL-10 and regulatory T-cell phenotypes [111]. Unfortunately, clinical studies using the clinically available S1P inhibitor, fingolimod, have been unsuccessful thus far [112,113]. Adaptive immune response Effector (CD4þ and CD8þ) and regulatory (CD4þCD25þFoxP3þ) T cells are important IRI and subsequent tissue recovery. Effector Tcells can remain in the kidney following IRI, function as memory T-cells [114], and further influence the development of chronic kidney disease and future adaptive immunological response to solid organ transplantation.

12

1. Ischemia-reperfusion injury

Early in the IRI injury, CD4þ T-cells are the first to be recruited and have been shown to influence the severity of IRI, with less hepatic injury in CD4þ T-cell deficient mice [115] and also influence neutrophil recruitment following hepatic IRI [116]. Both Th1 and Th2 CD4þ Tcell subsets are seen in renal parenchyma following IRI and have been shown to be dependent on IL16 [117] CD28-B7-1 (T-cell to endothelial cell) expression [118]. Inhibition of Th1 by blockade of the CCR5 receptor [119] or CXCR3 [120] protected murine kidney following IRI. Regulatory T-cells (Tregs) derived from either natural tolerance (self-tolerance to peripherally sampled antigens) or induced (exposure to antigens primed in the context of costimulation) are important in IRI. Worse renal IRI is seen with administration of anti-CD25 antibody, which depletes Tregs [121], and adoptive transfer of third party Tregs up to 24 h postinjury has shown benefit in animal models [122]. B lymphocytes are the latest to join in the inflammatory milieu and have been shown to have varying effects in renal IRI, ranging from protective [115,123,124] to impairing repair processes [125]. Although both T- and B- cell traditionally have been viewed as antagonists in inflammation and tissue injury, the presence of regulatory cells and participation in reparation processes are important given the depletion of both Tand B- cell confers no protection against renal IRI in mice [124].

Ischemia-reperfusion injury in acute kidney injury and transplantation Renal ischemia perfusion injury is a wellstudied (a prototypical example of for IRI in other organ systems) and will be used to highlight the current state research and future directions. The kidney is a complex organ with high metabolic demanddof which, the vascular endothelium and proximal renal tubular epithelial cells are the most metabolic active and

susceptible to ischemia and IRI. Common clinical scenarios at which IRI injury may cause acute kidney injury (AKI) include hypotensive and shock states (e.g., septic, cardiogenic, hemorrhagic shock), surgery (e.g., aortic clamping in cardiac surgery, abdominal vascular surgery), and delayed graft function (DGF) in the transplantation setting. In cases of severe AKI, the patient may need to be initiated on acute hemodialysis support, which is associated with increased in-hospital mortality, long-term risk of chronic kidney disease, and morbidity. Acute kidney injury is an important clinical problem, with an estimated one in five hospital admissions in developed countries complicated by acute kidney injury [126]. In 2012e13, there were over 130,000 cases of AKI hospitalizations and 10% of these patients do not survive the admission [127]. This is consistent with previous findings of increased morbidity and mortality associated with any incident diagnosis of AKI, particularly those who required renal replacement therapy (dialysis) during the admission [126e131]. These patients were more likely to have prolonged hospital stay [127] and the survivors of AKI are at higher risk of developing chronic kidney disease (CKD) and conversely, patients with underlying CKD are more susceptible to developing AKI in response to various insults and etiologies [128e130]. Despite this, there are no proven therapies that modify or treat AKI outside current strategies of prevention and supportive care.

Acute kidney injury and delayed graft functionddefinitions and implications Acute kidney injury is defined when there is an abrupt deterioration or reduction of kidney function based on clinical and laboratory findings. There are several diagnostic criteria in the literature and the three most commonly cited are KDIGO, RIFLE, and AKIN criteria [131e133] and are summarized in Table 1.2.

Ischemia-reperfusion injury in acute kidney injury and transplantation

13

TABLE 1.2 Acute Kidney Injury (AKI) Definitions. Summary of Key Criteria for AKI Definitions from the KDIGO Guidelines (The Kidney Disease: Improving Global Outcomes), AKIN Criteria (Acute Kidney Injury Network) and RIFLE Criteria (Risk, Injury, Failure, Loss of Kidney Function, and End-Stage Kidney Disease) [131e133]. Serum creatinine (SCr)

Urine output (UO)

Stage 1: Increase in SCr 26.5 mmol/L or 1.5e1.9 (from baseline) Stage 2: Increase SCr > 2e2.9 Stage 3: Increase SCr > 3, or SCr 354 mmol/L, or eGFR < 35 mL/min/1.73m2 in age < 18, or needing dialysis

2e2.9 Stage 3: Increase SCr > 3, or SCr 354 mmol/L, or needing dialysis

50% Failure: Increase SCr 3 or GFR decrease > 75% or SCr 354 mmol/L Loss: Persisting complete loss renal function > 4 weeks ESRD: More than 3 months

0.7, and bile/perfusate Naþ ratio >1.1 within 4 h of perfusion. In addition, Rocuronium levels during NEsLP were a good predictor for the development of primary non-function (PNF) after transplantation. Using a rat model, Op den Dries et al. investigated the preservation of the bile duct during NEsLP and found evidence that the bile duct epithelial cell function and morphology were better preserved in the perfused groups (DCD and no-DCD) compared to the SCS groups (DCD and no-DCD) [22]. In this study, a reperfusion model was used for liver assessment. During reperfusion, both SCS groups showed higher AST, ALT, and LDH levels than the perfused groups. In the two DCD groups, bile production was significantly higher in the grafts which were perfused compared to the ones which were cold stored. Gamma-glutamyl transferase (GGT) and LDH concentrations in the bile were used as biomarkers of biliary epithelial injury and were highest in the DCD þ SCS group. In addition, bile bicarbonate and pH were significantly higher in the perfusion groups, suggesting better biliary epithelial cells function. Hypothermic perfusion of liver grafts has also shown promising results in terms of protection against reperfusion injury [23]. In a rodent

173

transplantation model, Schlegel and colleagues determined that 1-h hypothermic oxygenated perfusion (HOPE) after 30 min of warm ischemia and 4 h of cold storage resulted in lower liver enzyme release (AST and ALT), higher Quick and Factor V serum levels, and improved ATP levels. In addition, 4 weeks after transplantation, HOPE-treated organs showed no biliary injury and less activated myofibroblasts. In another study, the same group investigated the mitochondrial rate of respiration during HOPE perfusion and observed decreased metabolism of NADH and decreased production of CO2 [24]. In addition, electron transfer rates were almost inexistent after more than 90 min of perfusion. In the same study, the authors investigated if the presence of oxygen is necessary and could show that anoxic hypothermic perfusion failed to prevent reperfusion injury as opposed to HOPE treatment. Moreover, they found evidence that a higher portal pressure of 8 mmHg has detrimental effects to the sinusoidal endothelia and that a lower portal pressure of 3 mmHg is appropriate for the cold perfusion. The authors concluded that end-ischemic oxygenated hypothermic machine perfusion at low pressure hinders endothelial damage and that oxygen delivery downregulates mitochondrial activity before reperfusion. However, no correlation was made between perfusion parameters during HOPE and liver function during reperfusion. Op den Dries and colleagues also investigated the effects of hypothermic machine perfusion for the preservation of DCD grafts [25]. After 30 min of DCD, livers were preserved for 4 h either on ice or with dual hypothermic oxygenated perfusion (DHOPE), followed by 2 h of reperfusion to simulate transplantation. Dual hypothermic oxygenated machine perfusion is another cold perfusion preservation method, in which the liver graft is perfused through both the hepatic artery and portal vein. Hepatic artery flow was higher in the DHOPE group during reperfusion, while portal vein flow was comparable between

174

7. Assessment of extended criteria liver grafts during machine perfusion. How far can we go?

groups. Hepatic ATP content was increased during DHOPE, but decreased during SCS. In addition, ATP levels increased in both groups during reperfusion. The release of liver enzymes after reperfusion was lower in the DHOPE group. Bile production, markers of biliary epithelial injury (biliary LDH and GGT) and function (biliary pH, glucose, bicarbonate) and gene expression of the biliary epithelial transporters involved in the secretion of bicarbonate into the bile were similar between groups. However, histology revealed signs of ischemia-reperfusion injury and less arteriolonecrosis of the peribiliary vascular plexus in the DHOPE group compared to the SCS group. Due to the low metabolism of the liver during hypothermic perfusion, graft assessment is challenging. At this moment, no animal study could correlate any of the perfusion parameters with the function of the liver after preservation. However, a Belgian group presented a score based on different perfusion parameters that correlated with the WI injury of livers in a porcine model [26]. The study included 6 groups of WIT (0, 15, 30, 45, 60, 120 min) divided into three clusters (0e15 min, 30e45 min, and 60e120 min). After retrieval, livers were subjected to 4 h of DHOPE followed by 2 h of reperfusion to simulate transplantation. Using three biochemical parametersdAST, pH, and fatting acid-binding proteindand vascular resistance, the authors developed a damage index. When using the index, the three clusters were significantly separated. To validate the score, the authors correlated the morphological score after normothermic isolated perfusion with the duration of WI. An association was observed between the damage index and the morphological score, which implicates a correlation between the damage index and WI. If this score could be validated in further studies, it could represent an important tool to assess the WI injury, which could be used when deciding to accept or reject an organ for transplantation.

Clinical studies Hypothermic machine perfusion Hypothermic machine perfusion is a standard preservation method in kidney transplantation and more recently has also been implemented in the setting of liver transplantation. Graft assessment during hypothermic machine perfusion is very limited, but liver function seems to be positively influenced by cold perfusion. Guarrera and colleagues presented the first prospective case-controlled liver anoxic HMP trial in 2010, in which they compared graft function and survival of 20 liver grafts that underwent HMP for 3e7 h with a matched group of liver grafts preserved with SCS [27]. Recipients of grafts preserved with HMP showed significantly less early allograft dysfunction (EAD), lower serum injury markers and shorter mean hospital stay. In an attempt to correlate perfusion parameters with liver function posttransplantation, peak serum AST and ALT after transplant was correlated with perfusion AST, ALT, and LDH at 2 h of HMP. Both peak recipient AST and ALT were significantly correlated with perfusion AST, ALT, and LDH at 2 h of HMP (Table 7.1). Dutkowski and colleagues were the first to investigate the safety of using oxygenated HMP in extended criteria liver grafts [28]. They compared the results of 8 DCD grafts that were treated with HOPE with those of matched DBD liver grafts. The recipients of DCD grafts showed lower liver enzyme levels than the recipients of DBD grafts and no intrahepatic biliary strictures could be identified at 3 and 6 months posttransplantation. Perfusion parameters were not correlated with the liver function posttransplantation (Table 7.1). In a subsequent study, the same group compared the results of 25 HOPE-treated DCD livers with matched cold-stored DCD grafts from two other centers and matched DBD grafts [29]. The HOPE-treated group showed lower

175

Clinical studies

TABLE 7.1

Experience on Transplantation of Liver Grafts Subjected to Hypothermic Ex Situ Machine Perfusion.

Author

n; Donor type Year; Site

Guarrera et al. [27]

20; DBD

2010; United States

Perfusion CIT time (h) (h) 4.3

Parameters for monitoring HMP

Assessment parameters

9.4

Hepatic artery and portal vein pressure; perfusate AST, ALT, LDH, lactate, pO2, pCO2

Perfusate AST, ALT, LDH at 2 h of perfusion

Dutkowski 8; et al. [28] DCD

2014 2 Switzerland

4.6

Portal vein pressure; perfusate pH, pO2, pCO2

-

Dutkowski 25; et al. [29] DCD

2015; 2 Switzerland

5.3

Portal vein pressure and flow

-

Guarrera et al. [31]

31; ECD

2015; United States

3.8

9.3

Hepatic artery and portal vein pressure; perfusate electrolytes, AST, ALT, LDH, lactate

Perfusate AST, LDH at 2 h of perfusion; portal vein pressure

van Rijn et al. [32]

10; DCD

2017; 2.1 Netherlands

8.7

Hepatic artery and portal vein pressure, flow and resistance; perfusate ALT, lactate, glucose, TBARS

-

Dutkowski 50; et al. [33] DCD

2019; NR Switzerland

NR

NR

Perfusate level of mitochondrial complex 1 injury

Schlegel et al. [30]

50; DCD

2019; 2 Switzerland

4.4

Portal vein pressure

-

Patrono et al. [34]

10; DBD

2019; Italy

5.2

Hepatic artery and portal vein flow and resistance

-

3

NR, not reported; TBARSs, thiobarbituric acid reactive substances.

liver transaminase levels and less EAD than the unperfused DCD grafts. In addition, PNF rate in the HOPE group was 0% compared to 6% in the unperfused group. The extrahepatic biliary complication rate was similar in both groups, but 1-year intrahepatic cholangiopathy was significantly lower in the perfused group. No graft loss was reported in the HOPE-treated group compared to 18% graft loss in the nonperfused group. When compared to the matched DBD group, the perfused group showed no significant differences in terms of reperfusion injury, graft function and survival, and biliary complications. In addition, no perfusion parameters were correlated with the liver function posttransplantation (Table 7.1). More recently, the Zurich group compared the results of 50

HOPE-treated DCD grafts with 50 matched SCS DBD grafts from the same centre and 50 matched SCS DCD grafts from a different centre. HOPE-treated DCD grafts showed less non-tumor-related graft loss and a higher 5-year graft survival rate compared to the SCS DCD grafts [30]. Peak 7-day ALT, renal replacement requirement and length of intensive care unit stay were similar in all groups. Perfusion parameters were not correlated with posttransplant graft function (Table 7.1). Guarrera and colleagues investigated the benefits of cold perfusion in ECD livers that were initially declined for transplantation [31]. He could demonstrate that, compared to a matched group of ECD livers that were preserved with SCS, the perfused group showed significantly

176

7. Assessment of extended criteria liver grafts during machine perfusion. How far can we go?

less biliary complications and a shorter mean hospital stay. EAD and 1-year survival rates were similar in the two groups. The authors attempted to find a correlation between the perfusion parameters and the liver function posttransplantation by plotting the peak serum AST of the recipient against the perfusate AST, ALT, LDH and lactate at 2 h of perfusion. A strong correlation was found between peak serum AST in the recipient and 2 h AST and LDH in the perfusate. Two cases in which the 2 h perfusate was the highest also presented high portal vein pressure over 7 mmHG during HMP. One of them developed PNF, and the other one was unstable upon reperfusion. Authors concluded that high 2 h perfusate AST and elevated portal vein pressure could predict imminent severe reperfusion injury [31] (Table 7.1). The Groningen group investigated the advantages of end-ischemic dual hypothermic oxygenated machine perfusion in DCD liver grafts [32]. Results of 10 DHOPE-preserved grafts were compared with matched DCD liver grafts that underwent SCS. Patient survival was similar in both groups, but graft survival was higher in the perfused group. Peak serum ALT and bilirubin on postoperative day 7 were significantly lower in the DHOPE group. In addition, DHOPE treatment resulted in a restoration of ATP levels and reduced reperfusion injury. Hepatic ATP levels at the end of the perfusion were significantly higher than in grafts preserved with SCS and comparable to hepatic ATP levels after reperfusion. No functional assessment of the liver was performed during perfusion (Table 7.1). More recently, an Italian group published their experience with cold perfusion in DBD grafts [34]. 25 grafts were subjected to HMP before transplantation (23 grafts received DHOPE perfusion and 2 grafts received HOPE perfusion) and compared to a matched group of DBD livers that were preserved with SCS. The perfused grafts had a lower postreperfusion syndrome (PRS) rate and less stage 2-3 acute kidney injury.

Also, the perfused group had lower peak serum transaminases and less early allograft dysfunction. Moreover, the authors found a correlation between the decrease of hepatic artery resistance throughout perfusion and the development of EAD after transplantation (Table 7.1). Quality and viability assessment during HMP remains sparse; but, there have been a few perfusion parameters that were correlated with graft function. Guarrera’s group could correlate peak recipient AST and ALT with 2-h effluent AST, ALT, and LDH [27,31]. In a recent study, 50 liver grafts that underwent HOPE treatment before transplantation were monitored by fluorometric analysis of released mitochondrial flavoproteins [33]. Measurement of mitochondrial flavoprotein release in machine perfusate correlated strongly with lactate clearance and coagulation factors at day 1 and 2 after transplantation. Even if currently only limited graft assessment is possible during hypothermic perfusion, several studies could prove the superiority of HMP over SCS. Moreover, its relative simplicity and lower costs compared to NEsLP makes it a valuable preservation method. Future studies are to investigate which other parameters could be assessed during HMP./t

Controlled oxygenated rewarming Recent data suggest that abrupt rewarming after cold preservation leads to reperfusion damage and possibly inferior graft function. Controlled oxygenated gradual rewarming (COR) might prevent this type of graft injury. The Minor group presented the first clinical results of grafts that had been subjected to slow controlled oxygenated rewarming before transplantation [35]. The small series of patients was compared to a matched cohort of historical patients. The COR group showed lower peak serum transaminases, and 6-month graft and patient survival was 100%. Graft assessment during COR perfusion has not been established yet. However, in this study, glucose perfusate concentrations were

Clinical studies

found to correlate with postoperative graft functiondlivers with subsequently inferior function showed a higher glucose release during perfusion. The authors argue that in grafts with more injured hepatocytes, glycogenolysis prevailed over the resumption of glycogen synthesis. In a follow-up study, the same group showed data from 15 patients that had received an organ after COR treatment [36]. The authors could correlate AST levels during perfusion at 20
C after 120 min with peak AST levels after transplantation. AST levels during the hypothermic perfusion period did not correlate with the AST levels posttransplantation.

Normothermic ex situ liver perfusion Normothermic liver perfusion maintains the grafts metabolically active, which allows for assessment before transplantation. When discussing warm perfusion and its results in DCD patients, the differences in protocols in different countries have to be considered. Most European countries do not allow giving heparin to the donor, while North American protocols include full heparinization of the donors. One of the first studies on discarded human livers showed that NEsLP adequately preserved liver parenchyma and did not increase the biliary epithelial cell loss [37]. In this study, for the assessment of hepatic injury and function, the authors used perfusate transaminases, lactate, glucose, pH, bicarbonate, and bile production and composition. In a follow-up study, the Groningen group identified bile production as a marker of liver graft viability [38]. Higher cumulative bile production correlated with the lower release of transaminases and potassium in the perfusate, normalization of glucose and lactate levels, and higher secretion of bilirubin, all markers of a better hepatobiliary function. The same group investigated a few years ago the activation of coagulation and fibrinolysis during NEsLP of 12 human discarded livers

177

[39]. They investigated prothrombin fragment F1þF2, D-dimer, plasmin-antiplasmin complex, tissue plasminogen activator, and plasminogen activator inhibitor-1 in perfusion fluid and concluded that warm perfusion actives fibrinolysis, but not coagulation. In addition, they used ALT levels in the perfusate as a marker of hepatocellular I/R injury and could correlate the concentration of tissue plasminogen activator and D-dimer with ALT perfusate concentration. The authors identified a high concentration of D-Dimer early after the start of warm perfusion as a marker of severe I/R injury and a predictor for poorer graft function. In a study by Vogel et al., the feasibility of extended warm perfusion was tested in 13 discarded human grafts, which were perfused for 24 h [40]. They could show that 24-h perfusion was possible, which could potentially provide extended criteria grafts with time for recovery and graft treatment. In addition, bile production during perfusion correlated with the histological grading of the liver postperfusion. Considering the promising results of warm perfusion in animal studies and with the discarded human livers, several clinical trials were started. In 2014, the group of Peter Friend reported a phase I clinical trial in the United Kingdom, which included 20 patients who received organs after NEsLP with the Metra Device [41]. As assessment parameters during perfusion, the authors report monitoring pH, bile production, and hepatic arterial and portal venous flows (Table 7.2). Outcomes of these patients were compared to a matched SCS control group. This study demonstrated that NEsLP is feasible and safe in the clinical setting. In addition, patients in the NEsLP group had significantly lower peak AST levels and less EAD compared with the SCS group. In this study, the perfusate was based on gelofusine and packed red blood cells. The first North American results on NEsLP in a clinical setting were reported by the Toronto group. Perfusions performed at the Toronto

178 TABLE 7.2

7. Assessment of extended criteria liver grafts during machine perfusion. How far can we go?

Experience on Transplantation of Liver Grafts Subjected to Normothermic Ex Situ Machine Perfusion.

Author

n; Donor type

Year; Site

Perfusion time (h) CIT (h)

Ravikumar et al. [41]

20; DBD and DCD

2016; UK

9.3

NR

Hepatic artery and portal vein flow; perfusate pH; bile production

Selzner et al. [42]

10; DBD and DCD

2016; Canada

8

NR

Hepatic artery and portal vein flow and pressure; perfusate pH, lactate, AST, ALT, ALP, bilirubin levels; bile production

Bral et al. [43]

10; DBD and DCD

2017; Canada

11.5

2.8

Hepatic artery and portal vein flow; perfusate pH, lactate, AST, ALT, bilirubin levels; bile production

Perera et al. [44]

1; DCD

2016; UK

6.9

7

Hepatic artery and portal vein flow; perfusate blood gas; bile production

Mergental et al. [45]

6; DBD and DCD

2016; UK

6.1

7.4

Hepatic artery and portal vein flow; perfusate lactate; bile production

Angelico et al. [46]

6; DBD and DCD

2016; UK

8.8

1.5

Hepatic artery and portal vein flow and pressure; perfusate pH and blood gas; bile production

Watson et al. [47]

12; DBD and DCD

2017; UK

4.7

7.1

Perfusate pH, lactate, glucose, ALT; bile production

Nasralla et al. [48]

121; DBD and DCD

2018; UK

9.1

2.1

Hepatic artery and portal vein flow; perfusate pH, lactate; bile production

Laing et al. [50]

To be reported

2017; UK

To be To be reported reported

Hepatic artery and portal vein flow; perfusate pH, lactate; bile production; glucose metabolism

Watson et al. [55]

47; DBD and DCD

2018; UK

NR

6.8

Bile pH, glucose concentration; perfusate pH, glucose, lactate, ALT

Ghinolfi et al. [60]

10; DBD

2018; Italy

4.2

4.1

Hepatic artery and portal vein flow; perfusate pH, lactate, glucose; bile production

Liu et al. [61]

21; DBD and DCD

2020; United States

5

3.2

Hepatic artery and portal vein flow; perfusate pH, lactate, glucose; bile production

Assessment during normothermic machine perfusion

NR, not reported.

General Hospital used the same Metra Device as the UK group; however, the perfusate was based on Steen solution and packed red blood cells [42]. In this study, outcomes of 10 patients who received a liver graft subjected to NEsLP were compared to a matched 1:3 SCS group. The results of this pilot study demonstrated that NEsLP with Steen solution is safe and well tolerated. In addition, the NEsLP group had lower transaminase levels on postoperative days 1e3, and there

was no difference in posttransplant intensive care unit and hospital stay. Our group perfused in total 34 livers; 15 were from HBD and 19 from DCD donors (data not published). Of these, 25 were transplanted and 9 livers were declined for transplantation due to severe acidosis despite the administration of high doses of bicarbonate and high lactate and glucose levels after several hours of perfusion. Other criteria that were considered for accepting a graft for transplantation include

Clinical studies

portal vein and hepatic arterial flow, bile production, and stability of perfusion. From the 25 transplanted patients, one developed nonanastomotic biliary stricture and one had primary nonfunction. The patient who developed primary nonfunction received an organ that required increased levels of bicarbonate, and even if lactate levels decreased initially quickly, it remained above 3.5 mmol/L at all times. From our experience, a low portal vein flow or arterial flow result in poorer outcomes, therefore portal vein flow over 800 mL/min and arterial flow over 150 mL/min are required for a graft to be considered for transplantation. In addition, lactate clearance is of paramount importance, and lactate levels have to remain below 2.5 mmol/L for at least 1 h, before any decision regarding acceptance can be taken (Fig. 7.1, Table 7.2). Glucose decrease and bile production are also considered when deciding whether a graft will be accepted for transplantation or not. During perfusion, pH levels are constantly corrected, and the necessity of high amounts of bicarbonate to maintain a

FIGURE 7.1

179

physiological pH level is unfavorable. After performing more than 30 perfusions, we have also concluded that a perfusion of at least 4 h is required before any decision can be taken. Another clinical trial was performed in Edmonton, Canada, but this group used the Metra Device with a perfusate based on Gelofusine and packed red blood cells, similar to the Oxford trial [43]. Results from the 10 liver grafts that were perfused before transplantation were compared to a 1:3 matched SCS group. All perfused organs functioned well and recipients of organs from both groups had similar AST, bilirubin, INR, and lactate levels posttransplantation. For the assessment of the perfusion, lactate clearance, bile output, and portal vein and hepatic artery flow were monitored (Table 7.2). In 2015, the Birmingham group was the first one to use NEsLP to recover a graft that had been declined for transplantation [44]. The liver was successfully used for transplantation and at 15-month posttransplantation the patient was doing well without signs of biliary strictures

Protocols for graft assessment during normothermic ex situ liver perfusion.

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7. Assessment of extended criteria liver grafts during machine perfusion. How far can we go?

(Table 7.2). The same group presented 1 year later data on 6 organs that had been initially declined for transplantation; after NEsLP, 5 of them were successfully rescued and could be transplanted [45]. For organ viability, authors assessed perfusate lactate, stability of arterial and portal venous flow, bile production, and homogenous graft perfusion. At a median follow-up of 7 months (range 6e19 months), all recipients were doing well, with normalized liver tests (Table 7.2). Another UK study from the Birmingham and Oxford Universities investigated the influence of NEsLP on the postreperfusion hemodynamics [46]. Results from six patients receiving grafts after NEsLP perfusion were compared with 12 matched patients receiving SCS grafts. Postreperfusion syndrome was defined as a drop of more than 30% in the mean arterial pressure, lasting for 1 min or longer within the first 5 min from graft reperfusion. No patient in the NEsLP group developed a PRS, compared to two cases in the SCS group. In addition, the machine perfusion group required less vasopressor infusion and blood product transfusion after graft reperfusion. For graft assessment during NMP, hepatic artery and portal vein flow and pressure, perfusate pH and blood gas analysis and bile production were monitored (Table 7.2). Watson and colleagues transplanted 12 extended criteria liver grafts that had been initially rejected for transplantation after using normothermic warm perfusion for viability assessment [47]. Grafts were evaluated based on changes in perfusate lactate, glucose, ALT, and pH levels (Table 7.2). Half of the grafts were perfused at high perfusate oxygen tensions (group 1), and the other half at near-physiologic oxygen tensions (group 2). Five of the patients in group 1 were diagnosed with postreperfusion syndrome and one had primary nonfunction. In group 2, all reperfusions were uneventful. Three of the twelve recipients developed cholangiopathy, and these grafts were unable to produce alkali bile during warm perfusion.

Recently, data from the first randomized trial comparing machine perfusion with static cold storage was published [48]. The UK group included in the study DBD and DCD livers from seven different centers, in four European countries. Patients who received a liver from the perfused group showed a significantly lower peak AST and lower risk for EAD; patient and graft survival did not differ significantly between groups. For graft assessment during perfusion, authors used hepatic artery and portal vein flow, perfusate pH, lactate clearance, and bile production (Fig. 7.1, Table 7.2). Despite no or minimal bile production during perfusion, 18 livers were transplanted and 17 of them functioned after transplantation. No correlation could be identified between bile production and posttransplant liver function or development of nonanastomotic biliary strictures. Acidosis could be correlated with poor outcome; one liver that had a lactate > 4 mmol/L for the duration of the machine perfusion developed PNF after transplant. Subgroup analysis showed that machine preserved DCD livers had a better outcome than both DCD and DBD cold-stored livers. In addition, fewer organs were discarded in the machine perfused group, which suggests that machine perfusion successfully increases organ utilization. Based on a series of 12 discarded human livers, the Birmingham group proposed a series of viability criteria for the acceptance of grafts for transplantation [49]. They identified a series of major and minor criteria assessed at 120 min after the perfusion was started and decided that a viable graft has to fulfill at least one major criterion and two minor criteria. Major criteria include lactate levels lower than 2.5 mmol/L and the presence of bile production and minor criteria include perfusate pH higher than 7.3, stable arterial flow higher than 150 mL/min, portal vein flow higher than 500 mL/min and homogenous perfusion of the liver (Fig. 7.1). These criteria were then adapted for the VITTAL clinical trial, a study protocol for viability testing

Clinical studies

and transplantation of extended criteria livers (VITTAL), which was meant to determine whether unused donor livers can be salvaged [50]. In this study, for a graft to be considered for transplantation, at least two of the following criteria had to be met within 4 h of perfusion: lactate levels lower than 2.5 mmol/L, evidence of bile production, perfusate pH higher than 7.3, evidence of glucose metabolism, stable arterial flow higher than 150 mL/min, portal vein flow higher than 500 mL/min and homogenous perfusion of the liver (Fig. 7.1, Table 7.2). Perfusion has to be run for at least 4 h, but no longer than 24 h. In this study, 31 livers underwent at least 4 h of NEsLP, and 22 of them were transplanted. When analyzing the perfusate, authors found that 52 metabolites distinguished EAD from non-EAD livers and 36 metabolites were different between patients who developed a PRS and those who did not [51]. This supports the idea that metabolic profiling could be used for viability assessment. In addition, the same study found that donor bile duct injury before machine perfusion and after reperfusion correlated with the development of biliary strictures [52]. The Birmingham group also found that transaminases levels correlate with lactate clearance during machine perfusion, but do not predict posttransplant complications such as EAD, PRS, or nonanastomotic biliary strictures [53]. Untargeted proteomic analysis of the perfusates was undertaken and a cluster of proteins was found, that could discriminate between transplantable and nontransplantable livers [54]. The Cambridge group presented recently their criteria for liver assessment during NEsLP [55]. In total, they perfused 47 grafts and transplanted 22 of them. In this series of perfusion, hepatic artery and portal vein flow did not correlate with the outcome or with other markers of hepatocellular injury. Lactate clearance has been used by several groups as viability marker [42,50]; however, in this study, a decrease in lactate was not found to be a predictive marker of liver viability, but

181

livers who did not clear lactate rapidly showed parenchymal injury. Although bile production did not correlate with liver function after transplantation, there was evidence that bile pH and glucose correlate with a stromal injury. The Cambridge criteria associated with a viable graft include maximum bile pH > 7.5, bile glucose concentration 10 mmol less than perfusate glucose, perfusate pH > 7.2 without more than 30 mL bicarbonate supplementation, falling glucose beyond 2 h or perfusate glucose under 10 mmol/L, peak lactate fall >4.4 mmol/L/kg/h, and ALT 70 years) was investigated by Ghinolfi and colleagues; 20 liver grafts were randomized to either SCS or NEsLP [60]. One patient in the perfusion group lost the graft due to hepatic artery thrombosis and one patient in the SCS group died with a functioning graft. There were no cases of PNF in any of the groups and postoperative transaminases and bilirubin peak were similar in both groups. During perfusion, hepatic artery and portal vein flow, perfusate pH, lactate and glucose and bile production were monitored. The authors also report a correlation between perfusate TNF-a, IL6, IL10 and lactate. Interestingly, electron microscopy analysis revealed less mitochondrial volume density and steatosis and more autophagic vacuoles in the perfused grafts, all signs of reduced ischemia reperfusion injury (Table 7.2). The Cleveland group recently investigated the safety of NEsLP using a customized perfusion device and a fresh frozen plasma (FFP)based perfusate [61]. Results from 21 liver grafts from both DCD and DBD donors subjected to warm perfusion were compared to a 1:4 matched non-perfused group. All perfused organs were successfully transplanted; one patient died at 8 months posttransplantation with a functioning graft. Recipients of NEsLP grafts had lower early allograft dysfunction rate and peak AST and ALT compared to the control group. During perfusion, portal vein and hepatic artery flow, perfusate pH, glucose and lactate levels, as well as bile production were monitored (Table 7.2). Overall, FFP based NEsLP proved safe and effective. Several parameters are available for graft assessment during normothermic ex situ machine perfusion. Most centers agree that the hepatic artery and portal vein flow are important markers of the quality of the graft. Considering

the amount of bicarbonate needed for maintaining a physiological pH and the ability to clear lactate are essential when deciding whether a graft is suitable for transplantation or not. The necessity of bile production is controversial, and there is no agreement on whether the level of transaminases in the perfusate plays an important role. Evidence of glucose metabolism is also an indicator of a viable graft and all authors agree that perfusion has to be run for at least several hours, before deciding whether a graft is suitable for transplantation or not.

Regional perfusion Abdominal regional perfusion is the newest approach in organ preservation and can be performed either hypothermic or normothermic. During regional perfusion, the abdominal organs are reperfused in situ with the donor’s own blood, using extracorporeal membrane oxygenation. At this moment, normothermic regional perfusion (NRP) is used for organ maintenance and not therapy. In uncontrolled donation after cardiac death (uDCD), cardiac arrest is unexpected, death is usually declared in the emergency room and NRP is usually started before the donor’s family has given consent. In controlled donation after cardiac death (cDCD), the timing of cannulation varies by country. Protocols in certain countries, such as Spain and the United States, allow heparinization and cannulation before the withdrawal of care. In the United Kingdom, cannulation can only be performed after the declaration of death. In uDCD, NRP is used to bridge the time from cardiac arrest until organ retrieval, which includes obtaining consent for donation, donor evaluation, and preparation. However, for cDCD, all preparations have been made and NRP is used for improving graft viability. Reports on the use of hypothermic regional perfusion (HRP) are very limited. A group

183

Future considerations

from Taiwan reported successful transplantation of a liver from a Maastricht category 4 DCD donor maintained with HRP after cardiac arrest and during organ recovery [58]. A group from Spain also reported the use of hypothermic regional perfusion with modest results. Graft assessment during HRP was not performed [59]. Normothermic regional perfusion was first reported in Spain in 1989 for uncontrolled DCD donors in kidney grafts [62]. The Barcelona Group initiated in 2002 a protocol to transplant liver grafts from Maastricht Category 2 DCD donors treated with NRP [63,64]. Their promising results encouraged also other Spanish and French centers to adopt an NRP protocol for uDCD donors [65,66]. All centers report promising results and at the moment, for uDCD donors, NRP appears to be the best preservation method. In the case of cDCD, the application of NRP is limited and most organs are still recovered with rapid in situ cold preservation. Last year, two multicenter studies have published data on outcomes in liver transplantation of cDCD grafts subjected to NRP postmortem. A Spanish multicenter study compared the results of 117 cDCD liver transplants performed without postmortem NRP with those of 95 cDCD liver transplants performed with postmortem NRP, with a median follow-up of 20 months [67]. Postoperative biliary complications and graft loss were significantly lower in the group with postmortem NRP. Two centers in the United Kingdom (Cambridge and Edinburgh) also recently published their experience with postmortem NRP [68]. They compared the outcomes of a group of 43 cDCD liver transplants performed with postmortem NRP with data of a contemporary cohort of 187 cDCD liver transplants performed without NRP. The NRP group showed less EAD and 30-day graft loss, no ischemic cholangiopathy, and fewer anastomotic strictures. Both studies showed clear data that postmortem NRP for cDCD grafts results in superior graft survival and less biliary

complications. Even if the assessment of liver grafts is not yet possible during NRP, the value of this preservation method is evident.

Future considerations The lack of organs with the result of patients dying on the waiting list remains the hard reality. This forces the transplant community to extend the inclusion criteria of organs and improve the preservation methods that are available at the moment. Inclusion criteria have become more permissive, and extended criteria liver grafts are being transplanted more and more. However, this also results in an increased number of complications, which further compels the transplant community to seek for ways of better preservation. Several machine preservation methods have been developed; however, it remains a matter of debate which one is best. Although hypothermic machine perfusion is relatively cheap, easier to control, and transport and has shown improved graft function compared to static cold storage, little assessment is possible due to the very low metabolism. One important issue with extended criteria grafts is to find ways to assess the organ beyond donor data. Machine perfusion can be the optimal platform for assessment and, if possible, treatment. Normothermic machine perfusion has demonstrated promising results both in an animal model and in human trials. However, it has higher costs and is a more resource-intensive preservation method. During warm perfusion, a series of parameters can be assessed and studies have shown that graft acceptance of extended criteria grafts is higher if grafts are subjected to NEsLP [48]. Performing warm perfusion during graft transportation is limited and can be extremely challenging. Therefore, for the time being, back to base warm perfusion is more achievable and could be adopted easier in a larger number of centers. For transportation, static cold storage

184

7. Assessment of extended criteria liver grafts during machine perfusion. How far can we go?

remains the standard. However, performing hypothermic machine perfusion during the transportation is a feasible option. Although graft assessment would be limited, it could offer better preservation during transportation and then warm perfusion could be performed back to base for assessment and, if necessary, treatment. Normothermic regional perfusion has also shown very good results both in uDCD and cDCD donors. This method reduces the warm ischemia time and, therefore, the ischemia injury. A combination of NRP and cold or warm perfusion is also an alternative. An Italian group reported their experience with four cases of uncontrolled DCD livers that were subjected to NRP and HOPE before transplantation [69]. NRP was conducted for a mean period of 318 min (252e360 min), and HOPE was performed for a mean period of 182.4 min (150e230 min). Livers were subjected to HOPE in case of expected long cold ischemia time. The final decision for transplantation was the macroscopic appearance of the liver and liver biopsy. No cases of PNF or ischemic cholangiopathy were present and all recipients showed good liver function posttransplantation. Both NRP and HOPE have shown promising results as preservation methods, so it is reasonable to assume that grafts would profit from a combination of the two methods. NRP could be used for graft preconditioning, and it is a good perfusion bridge between asystole and procurement, allowing a slower and less stressful organ procurement, which also minimizes the risk for accidents when retrieving the organs. HOPE is a relatively simple perfusion method that could be implemented during the transportation of grafts and while the recipient is being prepared for transplantation. The disadvantage of this combination is that no graft assessment can be performed. During NRP, graft assessment could be an option, but at this moment there is little data in this regard. This also implicates that a combination of NRP and warm perfusion might be the ideal

solution. A Spanish group presented data of one patient who received an organ that had been subjected to NRP for 211 min and NEsLP for 761 min [70]. The patient had a favorable clinical outcome without EAD after the transplant, AST/ALT peak was 1664/1015 IU/L and at 3 months’ follow-up, there were no signs of ischemic cholangiopathy. These results encourage our assumption, that a protocol that included NRP þ NEsLP could be the future of graft preservation for extended criteria liver grafts. The Groningen group presented interesting data on liver grafts that were subjected to DHOPE, then COR and then NMP before transplantation [71,72]. The study included livers that had been rejected for transplantation, but that were subjected to 1 h of cold perfusion for resuscitation, followed by controlled oxygenated rewarming and then warm perfusion for viability testing. Moreover, 11 livers from 16 were considered viable and were transplanted; at 3 months, patient and graft survival was 100%. Machine perfusion protocols increase the number of organs that are being used for transplantation. Both cold and warm perfusion have shown improved graft function compared to static cold storage. To further increase the number of organs considered for transplantation, graft assessment during machine perfusion has to be improved. The development of real-time measurement assays, such as the mitochondrial marker presented by the Zurich group [33], would greatly help in deciding whether a graft should be transplanted or not. A large number of proteins and metabolites are released during cold and warm perfusion in the perfusate [51,54]. Future studies need to verify if those could be correlated with the liver function posttransplantation. Even though the best markers for assessment of graft viability remain controversial, more and more centers are making tremendous efforts to improve machine perfusion and thereby graft preservation and assessment.

References

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lessons from the first 12 cases. Transplantation 2017; 101(5):1084e98. Nasralla D, Coussios CC, Mergental H, Akhtar MZ, Butler AJ, Ceresa CDL, et al. A randomized trial of normothermic preservation in liver transplantation. Nature 2018;557(7703):50e6. Mergental H, Stephenson BTF, Laing RW, Kirkham AJ, Neil DAH, Wallace LL, et al. Development of clinical criteria for functional assessment to predict primary nonfunction of high-risk livers using normothermic machine perfusion. Liver Transpl 2018;24(10): 1453e69. https://doi.org/10.1002/lt.25291. Laing RW, Mergental H, Yap C, Kirkham A, Whilku M, Barton D, et al. Viability testing and transplantation of marginal livers (VITTAL) using normothermic machine perfusion: study protocol for an open-label, non-randomised, prospective, single-arm trial. BMJ Open 2017;7(11):2017e017733. Attard JDW, Wallace L, Laing R, Boteon Y, Mergental H, Mirza D, Perera T, Afford S. Viability testing and transplantation of marginal donor livers (VITTAL) trial: metabolomics of ex vivo normothermically machine perfused livers discloses molecular signatures predictive of graft viability and post transplant outcomes [abstract]. Am J Transplant 2019; 19(Suppl. 3). Neil DHS, Laing R, Boteon Y, Attard S, Wallace L, Attard J, Perera T, Mirza D, Mergental H. Viability Testing and Transplantation of Marginal Donor Livers (VITTAL) Trial Outcomes: bile duct injury assessment during the normotehrmic machine perfusion of discarded livers [poster]. ILTS Annual Congres 2019. Boteon Y, Laing R, Attard J, Wallace L, Perera T, Mirza D, Afford S, Mergental H. Viability Testing and Transplantation of Marginal Donor Livers (VITTAL) Trial Outcomes: effectiveness of the lactate clearance-based viability criteria to discriminate hepatobiliary injury and post-transplant complications [poster]. ILTS Annual Congres 2019. Wallace LAJ, Boteon Y, Laing R, Yu J, Perera T, Mirza D, Mergental H, Afford S. Viability testing and transplantation of marginal donor livers (VITTAL) trial: NMP-L perfusate proteomics reveals biomarkers predictive of graft viability and post transplant complications ILTS Annual Congres. 2019. Watson CJE, Kosmoliaptsis V, Pley C, Randle L, Fear C, Crick K, et al. Observations on the ex-situ perfusion of livers for transplantation. Am J Transplant 2018;18(8): 2005e20. Schurink IJ dHJE, Minnee RC, Polak WG, Ijzermans JNM, van der Laan LJW, de Jonge J. Real-time metabolic function assessment of extended criteria donor liver grafts

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187 during normothermic machine perfusion. ILTS Annual Congres 2019. Matton APM, de Vries Y, Burlage LC, van Rijn R, Fujiyoshi M, de Meijer VE, et al. Biliary bicarbonate, pH, and glucose are suitable biomarkers of biliary viability during ex-situ normothermic machine perfusion of human donor livers. Transplantation 2019; 103(7):1405e13. Wang CC, Wang SH, Lin CC, Liu YW, Yong CC, Yang CH, et al. Liver transplantation from an uncontrolled non-heart-beating donor maintained on extracorporeal membrane oxygenation. Transplant Proc 2005;37(10):4331e3. Suarez F, Otero A, Solla M, Arnal F, Lorenzo MJ, Marini M, et al. Biliary complications after liver transplantation from maastricht category-2 non-heartbeating donors. Transplantation 2008;85(1):9e14. Ghinolfi D, Rreka E, De Tata V, Franzini M, Pezzati D, Fierabracci V, et al. Pilot, Open, randomized, prospective trial for normothermic machine perfusion evaluation in liver transplantation from older donors. Liver Transpl 2019;25(3):436e49. Liu Q, Hassan A, Pezzati D, Soliman B, Lomaglio L, Grady P, et al. Ex situ Liver machine perfusion: the impact of fresh frozen plasma. Liver Transpl 2020; 26(2):215e26. Sanchez-Fructuoso AI, Prats D, Torrente J, PerezContin MJ, Fernandez C, Alvarez J, et al. Renal transplantation from non-heart beating donors: a promising alternative to enlarge the donor pool. J Am Soc Nephrol 2000;11(2):350e8. Fondevila C, Hessheimer AJ, Ruiz A, Calatayud D, Ferrer J, Charco R, et al. Liver transplant using donors after unexpected cardiac death: novel preservation protocol and acceptance criteria. Am J Transplant 2007; 7(7):1849e55. Fondevila C, García-Valdecasas JC. Liver transplantation from donors after cardiac death. Digestive Liver Dis Suppl 2009;3(4):82e7. https://doi.org/10.1016/ S1594-5804(09)60031-0. Savier E, Dondero F, Vibert E, Eyraud D, Brisson H, Riou B, et al. First experience of liver transplantation with type 2 donation after cardiac death in France. Liver Transpl 2015;21(5):631e43.  Jimenez-Romero C, Manrique A, Calvo J, Caso O,  et al. Liver transplantaMarcacuzco A, García-Sesma A, tion using uncontrolled donors after circulatory death: a 10-year single-center experience. Transplantation 2019; 103(12):2497e505. https://doi.org/10.1097/tp.0000000 000002780. Hessheimer AJ, Coll E, Torres F, Ruiz P, Gastaca M, Rivas JI, et al. Normothermic regional perfusion vs. super-rapid recovery in controlled donation after

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circulatory death liver transplantation. J Hepatol 2019; 70(4):658e65. [68] Watson CJE, Hunt F, Messer S, Currie I, Large S, Sutherland A, et al. In-situ normothermic perfusion of livers in controlled circulatory death donation may prevent ischemic cholangiopathy and improve graft survival. Am J Transplant 2019;19(6):1745e58. [69] De Carlis R, Di Sandro S, Lauterio A, Ferla F, Dell’Acqua A, Zanierato M, et al. Successful donation after cardiac death liver transplants with prolonged warm ischemia time using normothermic regional perfusion. Liver Transpl 2017;23(2):166e73. [70] Pavel M-C, Reyner E, Fuster J, Garcia-Valdecasas JC. Liver transplantation from type II donation after cardiac death donor with normothermic regional perfusion and

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normothermic machine perfusion. Cir Esp 2018;96(8): 508e13. https://doi.org/10.1016/j.ciresp.2018.06.016. van Leeuwen OB dVY, Fujiyoshi M, Ubbink R, Pelgrim GJ, Werner JMJ, Reyntjens K, van den Berg AP, de Boer MT, de Kleine RHJ, de Meijer VE, Porte RJ. Increased and safe utilization of high-risk donor livers for trasnplantation after ex-situ resuscitation and assessment using sequential hypo- and normothermic machine perfusion ILTS Annual Congres. 2019. de Vries Y, Matton APM, Nijsten MWN, Werner MJM, van den Berg AP, de Boer MT, et al. Pretransplant sequential hypo- and normothermic machine perfusion of suboptimal livers donated after circulatory death using a hemoglobin-based oxygen carrier perfusion solution. Am J Transplant 2019;19(4):1202e11.

C H A P T E R

8

RNA interference in organ transplantation: next-generation medicine? Isabel M.A. Br€uggenwirth1, Paulo N. Martins2 1

Department of Surgery, Section of Hepato-Pancreato-Biliary Surgery and Liver Transplantation, University Medical Center Groningen, Groningen, The Netherlands; 2Department of Surgery, Division of Organ Transplantation, UMass Memorial Medical Center, University of Massachusetts, Worcester, MA, United States

Abbreviations ALI ALT AST CypD DA DC DCD DGF dsiRNA dsRNA ECD EVLP HIF-1 HLA HMGB-1 HMP IKK IL IRI LNP MD-2

acute lung injury alanine aminotransferase aspartate aminotransferase cyclophilin D deoxycholic acid dendritic cells donation after circulatory death delayed graft function dicer substrate RNA double-stranded RNA extended criteria donor ex vivo lung perfusion hypoxia inducible factor 1 human leukocyte antigen system high-mobility group box one hypothermic machine perfusion inhibitory kB kinase interleukin ischemia-reperfusion injury lipid nanoparticles myeloid differentiation protein-2

Organ Repair and Regeneration https://doi.org/10.1016/B978-0-12-819451-5.00008-1

MHC miRNA MPTP mRNA NALP3 NHE1 NMP PEI PHD1 PKCd RAGE RISC RNAi SCS SHARP-2 SHP-1 shRNA siRNA SLA TLR TNFa

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major histocompatibility microRNA mitochondrial permeability transition pore messenger RNA NACHT domain, leucine-rich repeat domain, and pyrin domain-containing protein-3 Naþ/Hþ exchanger 1 normothermic machine perfusion polyethyleneimine prolyl hydroxylase domain enzyme 1 protein kinase C delta receptor advances glycation end products RNA-induced silencing complex RNA interference static cold storage split- and hairy-related protein-2 Src homology region 2 domain-containing tyrosine phosphatase short hairpin RNA small interfering RNA swine leukocyte antigen toll-like receptor tumor necrosis factor-alpha

Copyright © 2021 Elsevier Inc. All rights reserved.

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8. RNA interference in organ transplantation: next-generation medicine?

Introduction Organ transplantation is often the only treatment for end-stage organ failure, such as for liver, kidney, lung, and heart. However, there is an ongoing discrepancy between transplant candidates and available organs. As a consequence, transplant centers are urged to use more suboptimal organs or organs from socalled extended criteria donors (ECD). ECD organs include grafts from donation after circulatory death (DCD) donors, organs that are elderly or steatotic, or biochemically compromised [1]. ECD and DCD organs are more vulnerable to the phenomenon known as ischemia-reperfusion injury (IRI). IRI occurs when the period of anoxia to a donor organ causes ischemic injury that is aggravated by the restoration of blood flow and reoxygenation (reperfusion) after implantation of the organ in the recipient [2]. Mitigation of IRI should drive the utilization of ECD organs to address the shortfall in donor grafts. Numerous studies have been conducted aimed to reduce IRI and improve outcomes after organ transplantation. More recently, the revolutionary discovery that specific gene silencing by RNA interference (RNAi) has the potential to inhibit genes related to IRI has led to increasing interest in this technique [3]. RNAi may open new avenues in the field of organ transplantation and improve outcomes, especially in suboptimal grafts. Ex situ machine perfusion has emerged as an exciting tool to recondition grafts before transplantation and reduce IRI. Good results have been shown after transplantation using machine perfusion over static cold storage (SCS) in all fields of organ transplantation [4e9]. Most studies have analyzed the effects of machine perfusion in suboptimal kidney and liver grafts, but increasing data is becoming available for other organs as well. Machine perfusion can be performed at temperatures ranging 4e37 C, whereas hypothermic (4 C) and normothermic

(37 C) machine perfusions are nowadays most commonly used [10]. Hypothermic machine perfusion (HMP) slows down the metabolism of the graft while providing oxygen, which restores energy levels. Normothermic machine perfusion (NMP) maintains the graft in a more physiological state, allowing viability assessment of the organ. More recently, it has become apparent that, besides the preservation of the graft, machine perfusion has the potential to “treat” the organ before transplantation [10]. Consequently, numerous treatment modalities during machine perfusion have been studied all aimed to reduce IRI and improve outcomes after transplantation. Targeted silencing of specific genes related to IRI using RNAi is one such modality [11]. Here, ex situ graft therapy has the advantage of reducing adverse effects related to systemic therapy. Another major advantage is that RNAi administered during ex situ machine perfusion is cheaper, because the dose is dependent on the organ weight instead of the full body weight. In addition, it is clinically more applicable, because RNAi treatment can take place after procurement, instead of treating the donor, which has regulatory implications in case of multiorgan procurement. In this chapter, we will present a brief history of RNAi and describe methods of inducing RNAi. We will present an overview of the studies that have been performed on RNAi in the field of organ transplantation, and we will focus on machine perfusion as a potential delivery platform for RNAi in organ transplantation.

Gene silencing with RNA interference In the early 1980s, it was revealed in Escherichia coli that small RNA molecules can bind to a complementary sequence in messenger RNA (mRNA) and inhibit translation [12]. More than a decade later, the American scientists Andrew Fire and Craig Mello demonstrated specific

Gene silencing with RNA interference

gene interference by double-stranded RNA (dsRNA) formation in roundworms. They realized that when sense and antisense RNA molecules meet, they bind to each other and form dsRNA. In particular, such dsRNA molecules can silence the gene carrying the same code as the particular RNA of interest. This seminal paper was the first to describe RNAi, and to coin its name accordingly [13]. RNAi was later observed in insects, animals, and humans. The natural function of RNAi appears to be the protection of the genome against invasion by mobile genetic elements, such as viruses, which produce aberrant RNA in the host cell when they become active. Specific mRNA degradation prevents the virus from replicating, although some viruses can overcome this process [14]. Despite the potency of RNAi, the technique could not yet be used for any therapeutic purposes in mammalian cells, owing to long dsRNA activating a “panic” response in eukaryotic cells [3]. This problem was overcome with the discovery of small interfering RNA (siRNA). It was demonstrated that after a long dsRNA enters the cytoplasm, an enzyme called “Dicer” cleaves the duplex into smaller 21e23 base-pairs, called siRNA. The siRNAs bind to an Argonaute protein (AGO), which is part of the multiprotein RNAinduced silencing complex (RISC), which unwinds the double-stranded siRNAs [15]. One strand of the siRNA is removed (“passenger strand”), after which the remaining strand is available to bind to mRNA target sequences according to the rules of base paring (A binds U, G binds C, and vice versa). An endoribonuclease then cleaves the mRNA, destroys it, or recruits accessory factors to regulate the target sequence in other ways. This way, the process of mRNA translation can be interrupted by siRNA, and expression of the targeted protein can be decreased (Fig. 8.1). In 2001, Elbashir et al. were the first to describe that synthetic siRNA was able to induce RNAi in mammalian cells [14].

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Until now, scientists have investigated that RNAi could be induced by four types of small RNA, including microRNA (miRNA), siRNA, short hairpin RNA (shRNA), and dicer substrate RNA (dsiRNA) [11]. These regulatory molecules differ in their biosynthetic pathway and the types of RISC they use. All four types of RNAi are currently in clinical trials to treat a wide spectrum of diseases. In 2006, the success of RNAi culminated after A. Fire and C. Mello won the Nobel Prize in Medicine [16]. Since then, RNAi has rapidly become one of the most powerful and widely used tools for the study of gene function. A series of important diseases have been targeted using siRNA-based therapies, including ocular and retinal disorders [17], several types of cancer [18,19], respiratory diseases [20,21], liver infection [22,23], and polyneuropathy [24]. In 2018, the federal drug agency approved Alnylam’s patisiran as the first siRNA drug targeting a protein that causes a rare, fatal form of amyloidosis [25]. At least six other RNAi therapeutics are already in phase III clinical trials, of which one targets p53 to reduce the incidence of delayed graft function (DGF) after kidney transplantation [25]. Even though the usage of siRNA seems a promising therapy in organ transplantation, some challenges need to be faced before widespread clinical application. The main hurdles associated with RNAi are related to stable delivery of siRNA to the target cells/organ, interaction of siRNA with blood components, immune responses, filtering by the liver or lungs, renal clearance, and undesired off-target effects.

Delivering strategies The utility of RNAi therapy relies on the effective delivery of the siRNA to the site of protein synthesis. Small-interfering RNA can be administered to the body via local or systemic delivery.

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8. RNA interference in organ transplantation: next-generation medicine?

FIGURE 8.1 Schematic illustration of the RNAi mechanism. Double-stranded RNA (dsRNA) molecule binds to a Dicer protein, which cleaves it into small interfering RNA (siRNA). These siRNAs bind to an Argonaute protein (AGO), which is part of the RNA-induced silencing complex (RISC). The RISC separates the siRNAs in two strands: the passenger strand (blue) is degraded, while the guide strand (orange) serves as a search probe, which links RISC to complementary RNA targets. After this recognition the target’s expression can be regulated through several different mechanisms.

Some advantages of local delivery are high bioavailability, reduced side-effects, and simple formulation. Local siRNA delivery has been used in clinical trials to mainly treat ocular, lung, and skin diseases [26]. However, most of the current preclinical and clinical development is focused on diseases that require systemic delivery of siRNA, such as cancer or viral infections. In 2003, the first successful systemic

delivery of siRNAs for therapeutic purposes involved the hydrodynamic injection of naked, unmodified siRNAs in mice to silence the death receptor Fas in the liver and prevent autoimmune hepatitis [27]. Currently, clinical trials using systemic delivery have mainly targeted diseases of the liver and kidney [26]. There are a number of factors that limit the therapeutic utility of RNAi [28]. First, the ability

RNAi in organ transplantation

of siRNA to enter the cytoplasm is limited as the molecules are water-soluble and cannot easily enter the cell via passive diffusion. In addition, naked siRNAs are extremely unstable in the physiological environment and are very easily and rapidly degraded by nucleases in the serum. Chemical modification techniques can improve serum stability and efficacy and increase the in vivo half-life and cellular uptake of siRNA. Common modification techniques include modifying the 20 -hydroxyl group on the ribosomal backbone, locking and unlocking the nucleic acid, and modifying the backbone. Another efficient way to deliver siRNA molecules into host cells is by using viral vectors [29]. The most commonly used vectors currently include lentiviral, retroviral, adenovirus, and adenovirusassociated virus vectors. Viral vectors have a high transduction efficiency and a high genesilencing effect in most target cells. The downside of viral vectors includes the low specificity of the virus carrier, which causes damage to other cells, the viral carrier itself can be randomly inserted into the host cell genome, causing activation of the host cell oncogenes, and viral vectors are associated with high costs. Besides viral vectors, nonviral vectors have been widely used to deliver siRNA [30]. Commonly used nonviral vectors include cationic lipids, polymers, dendrimers, and inorganic nanoparticles. In fact, the use of intravenously administered lipid nanoparticles and polymers resulted in a clinical proof of concept for systemic RNAi [31,32]. Although clinical progress has been made with these multicomponent systems, they are encumbered by the need for intravenous administration, and in the case of lipid nanoparticles, premedication with steroids to mitigate infusion-related reactions [31]. In the case of organ treatment before transplantation, ex situ machine perfusion can be an attractive approach for targeted siRNAdelivery to the organ [11]. It prevents filtration by other organs, enzymatic degradation of siRNA in the serum, and off-target effects. In

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addition, ethical and regulatory issues concerning donor treatment are overcome. siRNA may be added directly to the organ preservation solution, preventing the need for injection in the donor or recipient. Before transplantation, the organ may be flushed to remove the excess of circulating siRNA preventing off-target effects in the recipient.

RNAi in organ transplantation In this section, we will review the current status of the literature on RNAi in the field of liver, kidney, lung, and heart transplantation. Researchers have used RNAi to target genes specifically related to organ IRI to improve outcomes after transplantation. Fig. 8.2 shows major events in the history of RNAi since its first description in 1998. In addition, it shows the number of publications on RNAi in organ transplantation specifically.

Liver Studies using RNAi for the treatment of liver disease have accumulated since the beginning of this century. Numerous animal studies have been performed using RNAi for a broad range of purposes, such as to protect the liver from fulminant hepatitis [27], to inhibit hepatitis B or hepatitis C virus replication [33e35], to diminish hepatic damage after acute liver failure [36], to target liver fibrosis [37], or as a therapeutic intervention in hepatocellular carcinoma [38e40]. In addition, the silencing of genes associated with liver IRI may present a novel therapy in conditions associated with ischemic injuries, such as liver transplantation. IRI is an inevitable process that occurs during liver transplantation and represents the main cause of post-transplant graft dysfunction. The destructive effects of liver IRI mainly include direct cellular damage caused by ischemia and later hepatic injury resulting from inflammatory responses after reperfusion.

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8. RNA interference in organ transplantation: next-generation medicine?

FIGURE 8.2 Major events in the history of RNAi since its first description in 1998 and the number of publications on RNAi in liver, kidney, lung, and heart transplantation.

Owing to the liver’s unique anatomical architecture and availability of a large number of targets associated with liver IRI, as well as the development of some hepatic siRNA delivery methods, this organ has become an interesting model for the application of RNAi. Although many small animal studies use a hydrodynamic injection of siRNA, this approach is not practical for clinical use. Instead, a more selective and specific delivery method that allows for lower concentration siRNA to be used is needed for systemic delivery in humans. Lipid nanoparticles (LNP), for example, have attracted much interest as a safe therapeutic delivery method of siRNA to the liver, given its high hepatic retention after intravenous administration [41]. Proapoptotic proteins, like caspases, are known to be activated after reperfusion. The silencing of caspase-3 and caspase-8 was evaluated in a murine model of liver IRI [42]. Here, synthetic siRNA was delivered by injection via the portal vein 60 min before the induction of ischemia. Immunohistochemical analysis showed that 60% of hepatocytes efficiently took up siRNA. The siRNA-treated mice had lower serum aspartate aminotransferase (AST) levels, less infiltration of leukocytes, and better

preservation of the liver architecture compared to controls. Notably, 30% of animals given caspase-8 siRNA and 50% of animals given caspase-3 siRNA survived >30 days, whereas all of the control mice died within 5 days. On another note, Zhao et al. targeted high-mobility group box 1 (HMGB-1), an early mediator of injury and inflammation in IRI, in a mice model with 1-hour hepatic warm ischemia [43]. In vivo silencing was achieved through injection of siRNA through one of the lateral tail veins. The authors demonstrate reduced expression of HMGB-1 during liver ischemia, and improved liver function and a marked reduction in pathological liver damage after reperfusion. Another study assessed the effects of siRNA targeting RelB on liver IRI [44]. Systemic injection of RelB siRNA into mouse tail veins 24 h before liver ischemia effectively reduced the level of RelB. Silencing of RelB protected livers against IRI with reduced oxidative stress and a lower inflammatory response. Other studies have used shRNA, compared to siRNA, to target liver IRI. For example, the silencing of NALP3 (NACHT domain, leucinerich repeat domain, and pyrin domaincontaining protein-3) was achieved in murine

RNAi in organ transplantation

livers using shRNA against NALP3 administered via hydrodynamic injection 48 h before ischemia [45]. During IRI, NALP3 is pivotal in releasing cytokines interleukin (IL)-1b and IL18. Consequently, shRNA treatment resulted in decreased release of cytokines, decreased serum alanine aminotransferase levels (ALT), and decreased inflammatory cell infiltration. In a similar way, Hernandez-Alejandro and colleagues injected mice with tumor necrosis factor-alpha (TNFa) shRNA 48 h before inducing hepatic ischemia [46]. TNFa expression induced by IRI in the liver was significantly suppressed after shRNA treatment. After 6 h reperfusion, shRNA-treated mice show lower peak ALT and improved histology of liver parenchyma compared to controls. Schneider et al. aimed to silence the enzyme prolyl hydroxylase domain enzyme 1 (PHD1) in livers to protect hepatocytes against hypoxic damage and attenuate hepatic IRI [47]. PHD1 is an oxygen sensor that mediates cellular responses to changes in oxygen supply. Short hairpin RNA against PHD1 was administered via tail vein injection in mice 5 days before ischemia. Indeed, serum ALT levels were reduced in shRNA-treated mice, and silencing of PHD1 attenuated hepatocyte death induced by IRI. Although siRNA and shRNA can be applied to achieve similar functional outcomes, they are intrinsically different molecules. A main difference between the two is that shRNA can be continuously synthesized by the host cell, therefore, its effect should be much more durable [48]. In addition, higher doses are usually required for an effective knockdown in siRNA compared to shRNA, which can further contribute to off-target effects. As said, systemic administration of small RNA may cause off-target effects, which impedes clinical use. However, cationic liposomes have become an attractive delivery system for small RNA due to its low immunogenicity and ease of modification. A disadvantage is that this method is not cell-specific. Jiang et al. modified the liposome with galactose, which binds to

195

the asialoglycoprotein receptor that is expressed on the surface of hepatocytes. Their study reports for the first time the use of liver-specific liposome-based siRNA delivery to silence tolllike receptor (TLR) four in mice. Small-interfering RNA treatment efficiently inhibited the expression of TLR4 in the liver induced by IRI, which resulted in the prevention of liver IRI. Consequently, serum ALT was decreased, histology revealed reduced injury, oxidative damage was reduced, and lower levels of inflammatory cytokines were found [49]. RNAi in liver machine perfusion Over the last years, an increasing number of transplant centers worldwide are using machine perfusion to preserve liver grafts before transplantation. ECD livers in particular were shown to benefit from dynamic preservation by machine perfusion [4,6]. Small RNA delivery with machine perfusion might be beneficial for livers from ECD donors that would otherwise have been discarded. Recently, research from our group reported for the first time the use of siRNA during liver machine perfusion [50]. We demonstrated that Fas siRNA directly added to the perfusion solution can be successfully delivered to rat liver grafts during both hypothermic and normothermic machine perfusion. Transfection into hepatocytes was achieved by coating siRNA with LNPs. Small-interfering RNA-lipid complexes were delivered in the perfusion solution via portal vein cannulation, and distribution was assessed by fluorescent confocal microscopy. In addition, in a rat reperfusion model, we used siRNA together with LNPs to silence the apoptotic gene p53 [11]. Fig. 8.3 shows confocal microscopy images of p53 siRNA uptake by the liver during NMP. Uptake by the liver can be enhanced by encapsulating siRNA in LNPs or by using lipid-based delivery agents. Other lipid modifications to siRNA can drastically change distribution patterns in liver uptake, with the most lipophilic capable of uniformly liver tissue without interrupting the

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8. RNA interference in organ transplantation: next-generation medicine?

FIGURE 8.3 Alexa Fluoreconjugated p53 siRNA (1 mg/kg of the liver) is uptaken by rat hepatocytes during NMP with

Williams’ E medium. Scale represents 20 mm. (AeD) Untreated liver (no lipofectamine or siRNA) before machine perfusion. (A) Nuclei visualized in blue with DAPI, (B) siRNA in green, (C) cell membranes in red with wheat germ agglutinin conjugated to Alexa Fluor 647, and (D) with a merged image. (EeH) Liver treated with lipofectamine nanoparticles alone (no siRNA) and perfused for 2 h at 37 C. (IeL) Liver treated with lipofectamine with p53 siRNA and perfused for 2 h at 37 C. Adopted from Thijssen MF, Br€ uggenwirth IMA, Gillooly A, Khvorova A, Kowalik TF, Martins PN. Gene silencing with siRNA (RNA interference): a new therapeutic option during ex vivo machine liver perfusion preservation. Liver Transplant. 2019;25(1):140e151 with permission.

siRNA’s ability to interact with RISC machinery. In addition, it was shown that administering chemically stabilized siRNA with the preservation solution during machine perfusion leads to cellular uptake of the siRNA by the hepatocytes [51]. By selectively changing the siRNA backbone, a stable and targeted molecule can be synthesized specifically for delivery to liver grafts during the perfusion period [11].

Kidney The kidney could be an excellent target for RNAi therapy due to its unique characteristics of the urological system, which can lead to the rapid uptake of small RNA. In addition, the proximal tubular epithelium is a favorable target for synthetic small RNA accumulation after intravenous injection [52]. Over the years, there

RNAi in organ transplantation

have been reports of possible therapeutic benefits of RNAi in the treatment of several kidney diseases including primary hyperoxaluria [53e55], treatment of renal cell carcinoma [56e58], treatment of Wilms tumor [59], and suppression of secondary hyperparathyroidism [60]. In addition, it has been shown in animal models that RNAi has the potential to silence genes associated with IRI and improve outcomes after kidney transplantation.

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Several investigators have documented the importance of p53 activation in renal IRI. As such, Molitoris and colleagues report on the effects of p53 silencing to attenuate kidney IRI [52]. The authors present an illustrative figure in which they demonstrate kidney cell uptake of Cy3-labeled siRNA after intravenous injection (Fig. 8.4). There was rapid glomerular filtration of the Cy3-siRNA with subsequent proximal tubule brush border binding by proximal tubular

FIGURE 8.4 (A) Rapid filtration and uptake of fluorescence Cy3-siRNA in the living rat kidney as visualized by 2-photon microscopy. A high-resolution micrograph of the superficial renal cortex shows various landmarks after labeling with a 500 kD fluorescein dextran (green) and the nuclear dye Hoecshts 33,342 (cyan). (B) The nuclei of various epithelial and vascular cell types can be discerned; proximal tubules (PT); note the S1 segment (S1) opening up into the Bowman’s Space (Bow Sp) with adjacent Capillary Loops (CL). The 500 kD dextran shows the microvasculature seen between the PTs and outlines the CL within the glomerulus. The siRNA (seen in red) rapidly filters into the Bow Sp and down the S1 within seconds of infusion. (C) Within a minute after infusion, binding to the subapical region of PTs occurred. (D) Subapical endosomes can be seen in the lower left PT approximately 3-min postinjection. (E) The progression from binding to internalization is readily seen in PTs six0 min postinfusion. A distal tubule (DT) in the lower portion exhibits no uptake of the siRNA. (F) Degradation of the siRNA is apparent 24 h postinjection. There is a lack of residual fluorescence in PT or DTs. Bar ¼ 20 mm. Adopted from Molitoris BA, Dagher PC, Sandoval RM, Campos SB, Ashush H, Fridman E, et al. siRNA targeted to p53 attenuates ischemic and cisplatin-induced acute kidney injury. J Am Soc Nephrol. 2009;20(8):1754e1764 with permission through the Copyright Clearance Center (CCC).

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cells. Effective silencing of p53 was achieved after intravenous administration of siRNA targeting p53, and kidney injury was reduced. Moreover, the short duration of effect (70 years also increasing [10]. Adding to the complexity and risk of transplants is the growing use of combined organ transplants that now exceeds 4% of all heart transplant volume, particularly heart-kidney but also heart-liver [10]. As noted

MP and heart transplantation

earlier, the proportion of transplant recipients bridged with MCS has increased dramatically since 2007, especially in older recipients, but now appears to have stabilized at slightly more than 50% of transplants [10]. Increasingly MP is being utilized in high risk or marginal donors to both assess the viability of the procured organs, as well as to mitigate any further ischemic insult in an attempt to reduce rates of PGD, and early mortality in heart transplant recipients.

MP and heart transplantation Ex situ or machine perfusion has been of increasing interest in the area of organ preservation and may indeed prove to be the ideal strategy for organ management. The origins of isolated heart perfusion are found in research, originally employed by Carl Ludwig in 1866 in a frog heart, and subsequently established in mammalian hearts in 1895 by Langendorff [13], a version of his system still commonly used in cardiac research today. The advantages of MP are primarily derived from its ability to provide continuous oxygenated perfusion to the organ, thereby protecting it from ongoing ischemic injury. This ongoing perfusion of the heart allows for not only restoration of oxygen delivery, but also metabolic nutrients; as well as the removal of the potentially harmful by-products of metabolism. Furthermore, it provides both time and a platform to allow for the administration of cytoprotective and immunomodulatory agents [14], further regenerating and restoring the allograft. Finally, it allows for the assessment of biochemical function and viability of organs, crucial to allografts retrieved via a DCD pathway [15,16]. Despite these advantages, cold static storage (CSS) has remained the standard practice in most transplant units [17]. In CSS, the donor heart is examined and assessed in vivo before flushing of the heart with a cardioplegic preservation flush, rapid explantation, and storage in

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either saline or preservation solution at 4
C. The organ is then transferred to the recipient hospital for implantation. Procurement of hearts via this method generally allows for a tolerable period of total ischemia of between 4 and 6 h, after which, as discussed earlier, the organ deteriorates, with a higher risk of PGD and its associated recipient morbidity and mortality [2,10,12]. By restoring perfusion and its associated benefits, utilization of MP aims to offer a preservation modality permitting utilization of donor hearts that are currently considered too high risk for clinical transplantation, such as those with predicted ischemic times 6 h; and secondly, through ex situ support and evaluation improve the “quality” of all donor hearts irrespective of their donation pathway. Although to date there is only one widely used MP device, which utilizes normothermic machine perfusion (NMP); there has been a great deal of preclinical research and interest regarding the ideal conditions for machine perfusion, particularly NMP versus hypothermic machine perfusion (HMP). Both NMP and HMP rely on the restoration of oxygen and nutrient delivery; however, unlike NMP in HMP this is done with the heart in an arrested state. Under the profound hypothermic conditions (0e5
C) utilized in most studies for HMP, the heart’s metabolic requirements are low enough that the oxygen requirements can be met by low-flow administration of oxygenated crystalloid solutions [18]. The optimal perfusate composition and optimal pump parameters (pressure vs flow control, continuous vs pulsatile flow) have yet to be established for donor heart preservation, although it does appear, at least in NMP, that a whole blood perfusate provides superior preservation over either banked blood or modified CSS solutions [19,20]. In HMP, initial experience with modifications of standard CSS solutions (e.g., Celsior or the University of Wisconsin) was disappointing with the variable recovery of myocardial function as a result of nonhomogeneous tissue distribution of the perfusate, probably related to hyperkalemia induced coronary

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9. Repairing cardiac allografts on ex situ perfusion devices

vasoconstriction [21]. The use of normokalemic solutions, that more closely reflect the electrolyte composition of plasma, has been associated with improved tissue perfusion and functional recovery [21e23]. Despite these advances, there is still no clinically available HMP device (although there are several in preclinical phase and one in early human trials Table 9.1), and the use of such a device will be further complicated by the reduced ability for a functional or biochemical assessment of the viability of the heart in its arrested, hypothermic state. In contrast, the extensive preclinical investigation regarding NMP has resulted in its successful translation into clinical practice ([15,31e37], see Table 9.2). As noted earlier, the primary difference between HMP and NMP is the requirement for the blood-based, and preferably whole-blood, perfusate to permit delivery of sufficient oxygen to meet the metabolic demands of a normothermic beating heart [19,20]. Importantly, NMP allows for easier assessment of myocardial viability [16,38], although at present a tool for TABLE 9.1

true functional assessment of the hearts before implantation is not available for any device. Despite the lack of clarity regarding the optimal perfusate composition and pump parameters, reference ranges have been established for the TransMedics Organ Care System (OCS) to guide clinical management of donor allografts undergoing NMP. At present, the expertise required to operate the OCS device, as well as the requirement for donor blood collection (at a significant volume of 1.2e1.5 L), additional logistical difficulties associated with its transport, and the cost of the device have limited its widespread use. However, as evidence develops that this technology may both reduce rates of PGD as well as increasing the availability of donor organs, the overall costbenefit analysis will likely swing in its favor. As already noted, to date, the only machine clinically used for donor heart preservation is the TransMedics OCS, which is an NMP that utilizes a blood-based perfusate for perfusion of the donor heart. During donor organ procurement 1.2e1.5 L of donor blood is required to be collected, which

HMP Devices in the Preclinical Phase.

Device name

Animal studies

Human studies

XVIVO Heart Preservation Device (XVIVO Perfusion, Lund, Sweden)

Porcine DBD with 24 h nonischemic Heart transplantation using donor hearts hypothermic perfusion (NIHP) followed by preserved using NIHP [25]; 6 successful transplantation [24] clinical transplants performed to date [26,30a]. Planned international, multi-centre randomized controlled trial comparing NIHP to CSS, estimated enrollment of 202 participants. Due for completion in July 2022.

LifeCradle Heart Perfusion Organ Transport Systems Inc, Frisco, TX, USA

Porcine, non-DBD with 4 h continuous perfusion followed by transplantation [27]; Canine, DCD with 4 h continuous perfusion followed by transplantation [28]

HeartPort System (Modified Life Port System, Organ Recovery System, Itasca, IL, USA)

Porcine, non-DBD with 4 h of continuous perfusion followed by Langendorff assessment for 60 min [22,23]

28 rejected or not offered donor hearts recovered using CSS, antegrade perfusion or retrograde perfusion for 12 h followed by metabolic assessment [29]

Paragonix Sherpa Perfusion Cardiac Porcine, non-DBD with 12 h pulsatile Transport System (Paragonix perfusion followed by Langendorff Technologies, Baintree, MA, USA) assessment for 60 min [30] CSS, cold static storage; DBD, donation after brain death; DCD, donation after circulatory death; HMP, hypothermic machine perfusion.

MP and heart transplantation

TABLE 9.2

Studies and/or Units Utilizing NMP.

Study name (center)

Year

Donor N (OCS) criteria

PROTECT I (EU)

2006e07

PROTECT II (EU)

2007e08 20þ

Standard

PROCEED I (US)

2007e08

15

Standard

PROCEED II (US)

2011e13

62

Standard

Berlin

2009e13

20

Extended

Other German

2012e

13þ

Extended

Harefield (UK)

2013e

17þ

Extended

St Vincents (Aus)

2012e

10

Extended

EXPAND (US)

2015e

75þ

Extended

St Vincents (Aus)

2014e

50

DCD

Papworth (UK)

2015e

79b

DCD

Harefield (UK)

2015e

8þ

DCD

Wythenshawe (UK) 2017e

7

DCD

22

Manchester (UK)

2017e

3þ

Belgium (EU)a

2018e

3

Standard

DCD (unpublished) DCD

DCD, donation after circulatory death; NMP, normothermic machine perfusion; OCS, Organ Care System. a Belgium to date has performed three DCD transplants utilizing NRP and CSS without NMP. In the first two, the donors and recipients were colocated, and third, a pediatric donor was located nearby, with a short 2-h CIT [39,40]. b The Papworth DCD heart transplant program utilises two pathways; both DPP, and NRP with subsequent NMP, with current heart transplants performed via each pathway 57 and 22, respectively. DCD Heart Trial (USA), 2019, 47, DCD (unpublished).

after being passed through a leukocyte filter, is added to the pump reservoir with 500 mL of a proprietary priming solution containing metabolic nutrients (see Fig. 9.1). Before the heart being instrumented onto the OCS, a blood gas is performed, and any metabolic derangements corrected where possible. To facilitate perfusion on the OCS, the donor aorta is cannulated as well as the pulmonary artery. Both the superior and inferior vena cavae are closed to establish a “closed circuit” of the right heart and coronary perfusion, allowing for accurate measurement and monitoring of coronary flows. A vent is

217

introduced into the left ventricle (LV) via the open left atrium and secured. The heart is then connected to the OCS device and reperfusion commenced (see Fig. 9.2). A pulsatile pump delivers oxygenated blood from the reservoir to the ascending aorta. Upon closure of a competent aortic valve, the coronary arteries are perfused in an antegrade fashion, with deoxygenated blood returned to the right atrium via the coronary sinus. Deoxygenated blood is then ejected by the right ventricle via the return pulmonary artery cannula, passed through a low-resistance membrane oxygenator and returned to the reservoir. The heart is stabilized and maintained in a Langendorff, or nonworking configuration, with an unloaded but beating LV. The system also has the capability for defibrillation and temporary epicardial pacing if required. TransMedics’ recommended perfusion parameters are outlined in Table 9.3. Pump flow, as well as infusions of adrenaline and adenosine are used to control the coronary resistance and, in turn, the perfusion parameters. Once perfusion parameters on the device have been optimized, the potential allograft can be assessed for myocardial viability, primarily through the monitoring of arterial and venous lactates. Although in some allografts the initial lactate may be elevated, particularly in the case of DCD organs where they have been exposed to a period of warm ischemia, a viable heart will demonstrate a trend toward a reduction in the overall lactate levels, with an arterial to the venous differential, demonstrating an ability of the heart to extract lactate and utilize it as an energy source. According to the TransMedics guidelines, an allograft is viable if the overall lactate reduces to below 5 mmol/L. Multiple units have however demonstrated that in the presence of an appropriate downward lactate trend with evidence of extraction, it is not necessary for the overall lactate to be < 5 mmol/L, particularly in the setting of DCD organs where the starting lactate can often be > 10 mmol/L [31,35]. The

218

9. Repairing cardiac allografts on ex situ perfusion devices

FIGURE 9.1 Draining cannula in situ (A), Y-piece, and syringe with blood draining into a bag. A Y-piece with an attached 60 mL syringe is connected between the cannula and the collection bag (B,C). The collection bag with 25,000 U of heparin is placed on the floor and the donor placed in a Trendelenburg position to allow for rapid drainage of blood from the systemic venous system. If blood flow is not immediate, the syringe is used to siphon blood out of the right atrium and initiate the flow of blood into the bag [42].

sensitivity of lactate as a tool for assessing organ viability has been questioned in some studies; however, it remains the most practical tool available and, in many units, has been utilized with excellent outcomes [31,32,38]. The beating, normothermic state of the heart on the OCS

device, also permits for a degree of visual assessment, particularly of the RV, as well as adjunct assessments that have been performed in a limited number of cases, including the use of epicardial echocardiography and coronary angiography while on the OCS device [41].

MP and heart transplantation

219

FIGURE 9.2 Heart on OCS with ventricular pacing wires. The aortic cannula is deaired and then connected to the system with initial flows of 1 L/min. The superior vena cava is ligated and the inferior vena cava oversewn with a polypropylene 40 suture. A 16Fr left ventricular vent catheter (Medtronic, Minneapolis, MN) is placed through the open left atrium and mitral valve into the left ventricle for venting. This is held in position with a 3-0 polypropylene suture placed through the wall of the left atrium. The cannula draining the pulmonary artery is connected, and distension of the heart prevented by intermittent manual compression. Ventricular pacing wires are placed, and the heart defibrillated if necessary before the commencement of pacing at 80 bpm if the intrinsic rhythm is slower than this. Ao ¼ aorta; SVC ¼ superior vena cava; IVC ¼ inferior vena cava [42].

220 TABLE 9.3

9. Repairing cardiac allografts on ex situ perfusion devices

Normothermic Machine Perfusion (NMP) Target Parameters for the Initial Management of Organs.

Parameters

Starting dose and/or target

Pump flow (L/min)

0.9e1.2

Coronary flow (mL/ min)

650e850

Mean aortic pressure (mmHg)

60e90

Overall lactate

venous lactate

Manual/visual inspection

Exclude palpable disease and obvious wall motion abnormality or arrhythmia

Standard criteria brain-dead donors As discussed earlier, only one system utilizing normothermic perfusion is currently available for clinical use. The largest clinical trial conducted to date is the Proceed II Trial which utilized this system, the TransMedics OCS. It was designed as a prospective, open-label, multicenter, randomized noninferiority trial in 130 recipients of heart transplants from “standard criteria” donors [33]. The study compared 67 patients assigned to OCS allograft preservation with 63 patients whose hearts were preserved with standard CSS. Both groups had excellent outcomes with 94% and 95% 30-day survival rates, respectively, and no differences detected in rates of major adverse events. Although the trial achieved its primary goal of noninferiority, there was no advantage demonstrated of MP over CSS for these standard criteria donors. It is important to note, that the average age of the donors in each group was 35 and 34 years old, respectively, and the ischemic time for the allografts was well below 4 h, representing ideal conditions for successful transplantation [33]. One of the complexities in interpreting clinical studies and reports in transplantation is the lack of a consensus definition of a standard

versus marginal donor. There are many facilities who will accept donors up to the age of 50 of 55 years without considering these “marginal” donors, and similarly, due in large part to geographies, many units throughout the world are accustomed to ischemic times of >4 h in CSS, and would not consider this alone sufficient to categorize an organ as “marginal.” Recently published, the XVIVO hypothermic MP device has undergone a limited feasibility and safety trial in Europe [26,30a]. This was designed as a prospective, open-label, nonrandomized phase II trial in a single institution, with an additional three referring hospitals participating as donor sites. Although described as “standard” criteria for the study site, they adopted quite marginal donor criteria, including all accepted heart donors aged 30% macrosteatosis to be an indicator of significant postoperative morbidity, let alone a prognosticator of PNF. Outcomes with moderately steatotic grafts have varied widely over the years, and the early experience was dismal with high rates of EAD and PNF while recent experiences have demonstrated improved outcomes with more selective

250

11. Steatotic livers for transplantation: improving utilization of a prevalent resource

donor-recipient matching. In older studies, grafts with >30% macrosteatosis have been associated with higher rates of graft loss and EAD [6,18,20]. Similarly, recipients of these grafts have also been shown to have longer intensive care stays and higher transfusion requirements [21]. In addition, Croome et al. demonstrated a significantly higher incidence of postreperfusion syndrome, including intraoperative cardiac arrest, in recipients of moderately steatotic livers [22]. Although these grafts appear at increased risk of morbidity, there are also reports of successful outcomes in more recent series when extrinsic risk factors are minimized and recipients are carefully matched. Table 11.1, adapted from an excellent review by Linares et al. [23], demonstrates the range of reported outcomes with moderately steatotic grafts. Though these are largely single-center reports with small sample populations, they demonstrate that acceptable short and long-term outcomes can be achieved with the appropriate matching of donors to recipients [21,24e31].

Current trends in transplantation of steatotic livers Despite small sample populations, recent studies of liver transplant with moderate or severely steatotic grafts have demonstrated improved short and long-term outcomes if grafts are matched to an appropriate recipient and other modifiable risk factors are minimized. As an example, avoiding grafts from DCD donors or keeping CITs to a minimum tend to improve

TABLE 11.1

Study

Clinical studies and outcomes with transplantation of moderately steatotic livers. Sample size PNF

EAD

1-year graft survival

3-year patient survival

Croome et al (2019) [22]

96

4% (4/96)

76% (68/94)

79% (76/96)

73% (70/96)

Kron et al (2018) [40]

9

11% (1/9)

–

44% (4/9)

–

Outcomes with severe macrosteatosis

Chavin et al (2013) [26]

27

0% (0/27)

35% (10/27)

81% (22/27)

70% (19/27)

As with moderately steatotic grafts, livers with severe macrosteatosis (>60%) have traditionally been discarded. This is evidenced by early studies that demonstrated unacceptably high rates of PNF and EAD [26e29]. However, transplant centers have also reported acceptable outcomes when these grafts are used judiciously. McCormack et al. reported equivocal 60-day and 3-year survival in recipients of severely steatotic livers matched to a control group, though with higher rates of EAD, renal failure, and longer hospital stays [32]. Wong et al. reported excellent outcomes with 19 severely steatotic livers, including 0% EAD and PNF, as well as 94.7% 1- and 3-year overall survival [33]. Notably, recipients in their series were relatively healthy (median MELD 20), and grafts were all procured from brain dead donors with relatively short cold ischemic times (median 384 min).

Gabrielli et al (2012) [27]

6

0% (0/6)

–

100% (6/6)

100% (6/6)

De Graaf et al 7 (2012) [28]

0% (0/7)

29% (2/7)

100% (7/7)

–

Doyle et al (2010) [21]

0% (0/22)

–

82% (18/22)

70% (15/22)

Noujaim et al 6 (2009) [29]

0% (0/6)

0% (0/6)

50% (3/6)

50% (3/6)

Li et al (2009) [25]

23

4% (1/23)

22% (5/23)

91% (21/23)

–

Gao et al (2009) [30]

24

0% (0/24)

20.8% (5/24)

83% (20/24)

–

Burra et al (2009) [31]

11

0% (0/11)

–

82% (9/11)

82% (9/11)

Angele et al (2008) [24]

50

4% (2/50)

–

72% (36/50)

58% (29/50)*

* 5-year survival. Table

22

adapted from Linares et al. (23)

Current use of machine perfusion for steatotic livers

outcomes [33,34]. Jackson et al. performed a comprehensive analysis of recipient risk factors that affected outcomes with steatotic liver transplantation, demonstrating that first-time recipients with a MELD 15e34, without primary biliary cirrhosis, and not on life support before transplant as factors associated with equivalent mortality and graft loss to nonsteatotic livers [35]. In a related study, the same authors demonstrated decreasing discards and improving rates of mortality and graft loss with transplantation of moderate and severely steatotic livers from 2012 to 2017 compared to 2005e11 [8], indicating an increasing willingness of surgeons to use grafts with significant steatosis, as well as improved outcomes with careful donor and recipient selection.

Current use of machine perfusion for steatotic livers As machine perfusion has been increasingly adopted in the last decade, so too has the utility of steatotic grafts for transplant. Different perfusion modalities have unique costs and benefits. The two most commonly used modalities are normothermic (NMP) and hypothermic oxygenated perfusion (HOPE). Several groups have reported their clinical experience with perfusion of steatotic grafts.

Normothermic perfusion Two clinical studies included steatotic livers. The first is the widely publicized randomized clinical trial by Nasralla et al. [36] comparing the efficacy of continuous NMP (perfusion started at the donor hospital) to standard cold storage in a European cohort. Of 121 grafts that were preserved with NMP and subsequently transplanted, 29 (24%) have moderate or severe steatosis. It should be carefully noted that steatosis was assessed clinically by the retrieval surgeon and not with biopsy, which obfuscates the accuracy of assessment [37]. No further subgroup analysis

251

of these grafts is provided, limiting conclusions regarding the functional and graft outcomes of steatotic livers undergoing NMP. Interestingly, of the 16 grafts discarded in the NMP arm, 13 were described as steatotic. 8 of these livers were discarded for reasons relating to poor perfusion parameters or excessive steatosis. Ceresa et al. compared a back-to-base NMP protocol [38] to the continuous NMP arm used in the study by Nasralla et al. In this study, grafts were brought back to the recipient hospital under cold storage and subsequently underwent NMP before transplantation. Six grafts enrolled in the back-to-base arm demonstrated moderate or severe steatosis on final histology, of which one was discarded due to poor function during NMP. No individual recipient data are available but no significant differences in 1-year graft survival or postoperative complications were seen in the 31 transplanted grafts compared to the continuous NMP cohort. In a preclinical study, Liu et al. subjected 10 discarded grafts of varying steatosis to 24 h of NMP [39]. The majority of the grafts with mild macrosteatosis demonstrate adequate lactate clearance, bile production, and low vascular resistance. Notably, of the three grafts with moderate or severe macrosteatosis, bile production was relatively low and liver enzyme higher than the other grafts. Interestingly, there was no significant change in macrosteatosis in the cohort after 24 h of NMP. Lipid analysis demonstrated highand low-density lipoprotein (HDL, LDL) levels decrease significantly, while triglyceride levels increase significantly in the perfusate during NMP.

Hypothermic oxygenated perfusion Kron et al. describe in a single-institutional series of steatotic grafts the addition of HOPE compared to a cold storage [40]. In this clinical study (supported by evidence from rat liver transplant experiments), 6 steatotic grafts (range 20% e40% macrosteatosis) from DCD donors

252

11. Steatotic livers for transplantation: improving utilization of a prevalent resource

underwent end-ischemic HOPE and were successfully transplanted with 100% 1-year patient survival and no incidence of PNF. Comparatively, the control group of 12 steatotic grafts (range 20% e60% macrosteatosis) from brain dead donors were transplanted in standard fashion but demonstrated significantly worse outcomes, including 42% 1-year survival and 25% rate of PNF. Three patients in the control arm required retransplant. The addition of HOPE also resulted in significantly lower ALT, INR, and bilirubin trends in the postoperative period.

Conclusions and limitations from current studies Despite burgeoning evidence that steatotic livers will soon make up a significant portion of available grafts for transplant, little clinical or preclinical research is being directed toward categorizing and evaluating the function and outcomes of these organs during perfusion. Promising evidence from the investigators at the University of Zurich point to early success with transplantation of steatotic grafts, despite warm ischemic injury, after deployment of HOPE. Data from clinical studies of NMP that enroll steatotic grafts lack granularity needed to draw insightful conclusions, but these studies may warrant post hoc analysis.

Future therapies for repairing steatotic livers The application of machine perfusion for the resuscitation of steatotic grafts has demonstrated early success. However, most published clinical experience with perfusion of steatotic grafts is limited to