Diagnostic and Therapeutic Implications of Ex Vivo Lung Perfusion in Lung Transplantation: Potential Benefits and Inherent Limitations : Transplantation

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Diagnostic and Therapeutic Implications of Ex Vivo Lung Perfusion in Lung Transplantation: Potential Benefits and Inherent Limitations

Yu, Jing MD1; Zhang, Nan MD1; Zhang, Zhiyuan BMc1; Li, Yuping PhD2; Gao, Jiameng BMc1; Chen, Chang PhD2; Wen, Zongmei PhD1

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Transplantation 107(1):p 105-116, January 2023. | DOI: 10.1097/TP.0000000000004414
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Abstract

INTRODUCTION

Lung transplantation (LTx) remains the only ultimate therapeutic strategy for patients with end-stage lung disease. Despite tremendous advances made over the past few decades, LTx is frustratingly limited by numerous factors, including high morbidity and mortality, limited donor availability, and potential concerns about lung quality.1,2 In particular, a significant discrepancy exists between the increasing demand for LTx and the severe shortage of suitable donor lungs, which necessitates the usage of extended criteria donor (ECD) lungs.3,4 Continuous endeavors to maximize the donor pool by developing early efficient evaluation and intervention tactics for ECD lungs are ongoing.

Recently, Clavien and colleagues reported the successful transplantation of a discarded donor liver that underwent 3 d of ex situ normothermic machine perfusion, showing the great potential of machine perfusion in organ preservation.5 Advancements in machine perfusion technology have also rendered ex vivo lung perfusion (EVLP) a powerful tool in LTx for preserving and assessing donor lungs, fueling the continued enthusiasm among researchers in using ECD lungs.6 Various clinical trials have demonstrated the role of EVLP in expanding the donor pool and prolonging preservation time.7-9 EVLP allows normothermic perfusion and mechanical ventilation, maintaining physiological conditions similar to those in vivo, which facilitates more translational studies.10,11 Promisingly, EVLP displays great value in decreasing the risk of primary graft dysfunction (PGD).12,13 Despite these benefits, however, EVLP is not quite a technological panacea for LTx. Unfortunately, not all donor lungs are salvageable, and the potential adverse effects of EVLP should be addressed.14,15 In this review, we revisit clinical trials and examine relevant diagnostic and therapeutic interventions during EVLP. We also elaborate on the limitations of EVLP, providing a theoretical basis for further clinical practice of EVLP.

EVLP EVOLUTION

The first attempt at ex vivo organ preservation with oxygenated serum was performed as early as the 1930s.16 The canine lung perfusion model was explored in 1970, highlighting the potential role of an ex vivo perfusion system in assessing organ function.17 In 1987, a dynamic method of preservation for heart-lung transplantation was developed, which aimed to extend the preservation period of organs through a modified autoperfusion method to provide normothermic pulmonary and coronary blood flow.18 After decades of technological evolution, the contemporary EVLP device enables the continuous perfusion and ventilation of donor lungs in a normothermic environment. The first clinical case of donor lung evaluation by EVLP before LTx was reported by Steen and colleagues in 2001.19 In particular, they developed the revolutionary acellular perfusate Steen, which has an optimum colloid osmotic pressure.20 Thereafter, 2 commercially available EVLP devices, the XVIVO Perfusion AB system and the Organ Care System (OCS) lung system, were approved by the Food and Drug Administration in 2019, ushering in a new era in the field of LTx20 (Figure 1A).

F1
FIGURE 1.:
A, Timeline of major developments of EVLP. B, EVLP circuit of XPS XVIVO Perfusion AB system. The arrows denote the direction of flow. The red color denotes the outflow from the LA; the blue color denotes inflow to the PA. EVLP, ex vivo lung perfusion; FDA, food and drug administration; LA, left atrium; PA, pulmonary artery.

EVLP PROTOCOLS

Three EVLP protocols are currently available, including the Toronto, OCS, and Lund protocols.21 In addition to the device used, the perfusate composition, ventilatory setting, and other technical parameters vary. The OCS Lung system used in the OCS protocol is the only portable option and is mainly utilized for preservation instead of static cold storage (SCS); this system can generate pulsatile perfusion similar to pulmonary blood flow. The perfusate in the Toronto and Lund protocols is Steen-based, whereas the OCS protocol utilizes OCS or Perfadex. In the Lund and OCS protocols, red blood cells are added to the perfusate to maintain a hematocrit of approximately 20%, whereas the Toronto perfusate is acellular. A pulmonary arterial flow of 40% of cardiac output (CO) is set in the Toronto and OCS protocols, respectively, whereas 100% of CO is the preferred flow in the Lund protocol. Additionally, an open left atrium (LA) is present in the Lund and OCS protocols, whereas a closed LA (pressure of 3–5 mm Hg) is present in the Toronto protocol. The Toronto protocol utilizes the XVIVO Perfusion AB system, which is composed of a ventilator and a perfusion circuit. The circuit includes a reservoir, a centrifugal pump, a membrane oxygenator, a heat exchanger, and a leukocyte filter. Additionally, an organ chamber is specially designed for the placement of the donor lung. The continuous perfusate drains from the LA into the reservoir by gravity and is subsequently pushed into the hollow fiber oxygenator by the centrifugal pump, wherein the perfusate is deoxygenated. Finally, the deoxygenated perfusate is filtered to remove leukocytes before re-entering the pulmonary artery (PA)21 (Figure 1B). The median perfusion time in the Toronto protocol is recommended to be 4–6 h but no longer than 12 h.22 Because of higher CO, the perfusion time in the Lund protocol ranges from 1 to 2 h; however, the latter has been proven to be beneficial.23 In the OCS protocol, the perfusion time varies according to the duration of organ transportation; in a pilot study, the mean time was 5 h (range 3–10).24 Considering long-distance transportation, more studies are required to demonstrate the long-term feasibility of the OCS protocol.

POTENTIAL BENEFITS OF EVLP ACCORDING TO CLINICAL AND PRECLINICAL EVIDENCE

Clinical Evidence

Increasing the Donor Pool

Conventionally, >80% of donor lungs not considered suitable for LTx because of potential injury would be discarded. However, in the EVLP era, an increasing number of clinical trials have proven the safety and efficacy of EVLP in either ECD or standard lungs (Table 1). In 2008, a prospective landmark clinical trial was conducted by the Toronto lung transplant program.25 They demonstrated a similar rate of PGD (15% versus 30%, P = 0.11), duration of posttransplant mechanical ventilation, length of stay in the intensive care unit (ICU) and hospital, and survival rate at 1 y (80% versus 83.6%, P = 0.54) in high-risk donor lungs that had undergone EVLP and those that were conventionally eligible. Consequently, 87% (20/23) of high-risk donor lungs were used. Likewise, several clinical trials have demonstrated similar posttransplant outcomes between EVLP-treated ECD lungs and standard donor lungs.26-30 However, these studies were nonrandomized, and only ECD lungs were subjected to EVLP; thus, there could have been biased. A randomized and prospective study investigated the effect of EVLP on 80 recruited standard donor lungs.31 EVLP resulted in a lower incidence of PGD (5.7% versus 19.5%); however, because of sample size limitations, a significant difference was not achieved between the EVLP and non-EVLP groups (P = 0.10). Although the clinical trial demonstrated the safety of EVLP in standard donor lungs, a better-powered randomized study is required to confirm the superiority of EVLP.

TABLE 1. - Summary of clinical EVLP trials in LTx
Study and time Study design EVLP protocol Control group EVLP group Conversion to LT (%) Posttransplant results
Cypel et al 8 Prospective, nonrandomized Toronto SCS, n = 253 ECD, n = 50 50/58 (86%) Similar: PGD3 at 72 h, ECMO usage, time to extubation, ICU stay, hospital stay, mortality at 30 d, 1-y survival
Cypel et al 25 Prospective, nonrandomized Toronto SCS, n = 116 ECD, n = 20 20/23 (87%) Similar: PGD3 at 72 h, MV time, ICU stay, hospital stay, 1-y survival
Tikkanen et al 32 Retrospective Toronto SCS, n = 340 ECD, n = 63 63/73 (86%) Similar: Allograft survival and freedom from CLAD at 1, 3, and 5 y, FEV1%, 6-min walking distance, and acute rejection at 1 y
Aigner et al 27 Prospective, nonrandomized Toronto SCS, n = 119 ECD, n = 9 9/13 (69%) Similar: PGD3 at 72 h, MV time, ICU stay, hospital stay, 30-d survival
Warnecke et al 24 Single-arm, 2-center OCS N/A SCS, n = 12 12/12 (100%) All recipients showed a survival rate of 100% at 30 d.
Warnecke et al 33 Prospective, randomized OCS SCS, n = 169 SCS, n = 141 141/141 (100%) EVLP: Decreased total ischemic time, lower PGD3 at 72 h, 79.4% of patients met the primary endpoint
Nilsson et al 34 Prospective, nonrandomized, 2-center Lund SCS, n = 271 ECD, n = 61 54/61 (89%) Similar: FEV1.0% at 1 y, 5-y survival
aEVLP: Longer time to extubation, ICU stay
Valenza et al 7 Prospective, nonrandomized Lund SCS, n = 28 ECD, n = 7 7/8 (87%) Similar: PGD3 at 72 h, mortality at 30 d, overall survival
Boffini et al 13 Prospective, nonrandomized Toronto SCS, n = 28 ECD, n = 8 8/11 (73%) Similar: PGD3 at 72 h, ECMO usage
Wallinder et al 26 Prospective, nonrandomized Lund SCS, n = 47 ECD, n = 11 11/11 (100%) Similar: PGD3 at 72 h, hospital stay EVLP: Longer time to extubation, ICU stay
Sageet al 29 Prospective, nonrandomized Toronto SCS, n = 81 ECD, n = 31 31/32 (97%) Similar: PGD3 at 72 h, MV time, ICU stay, hospital stay, 1-y survival
Zhang et al 30 Prospective, nonrandomized Toronto SCS, n = 18 ECD, n = 9 9/9 (100%) Similar: FEV1% and FVC% at 3 and 24 mo/3-y survival EVLP: 33% developed PGD1 at 72 h
aControl: 11% developed PGD1, 6% PGD2, and 11% PGD3 at 72 h
Fisher et al 35 Prospective, nonrandomized, multicenter Lund SCS, n = 184 ECD, n = 18 18/53 (34%) Similar: PGD3 at 72 h, hospital stay; survival, infection, lung function and rejection at 12 moEVLP: Longer time of ICU stay, higher ECMO support and cost
Slama et al 31 Prospective, randomized Toronto SCS, n = 41 SCS, n = 35 35/39 (90%) Similar: PGD2 or PGD3 at 72 h, intubation time, ICU stay, hospital stay, 30-d survivalEVLP: Lower ECMO usage
Loor et al 9 Single-arm, multicenter OCS N/A ECD, n = 79 79/91 (87%) The rate of PGD3 at 72 h and mortality at 30 d was 44% and 99%, respectively
Fildes et al 28 Prospective, nonrandomized Lund SCS, n = 46 ECD, n = 9 9/9 (100%) Similar: Acute rejection, treated infection episodes up to 12 mo, ICU stay, hospital stay, 1-y mortality
CLAD, chronic lung allograft dysfunction; ECD, extended criteria donor; ECMO, extracorporeal membrane oxygenation; EVLP, ex vivo lung perfusion; FEV 1.0%, forced expiratory volume in 1 s; ICU, intensive care unit; LTx, lung transplantation; MV, mechanical ventilation; N/A, not applicable; OCS: Organ Care System; PGD1/2/3, grade 1/2/3 primary graft dysfunction; SCS, static cold storage.

The experience of using the OCS Lung device was first described by Warnecke et al in 2012.24 The survival rate was 100% among all 12 recipients at 30 d after LTx, demonstrating the safety of the OCS lung device used for organ preservation. To compare the beneficial effects of OCS with those of SCS, a prospective, randomized, controlled, multicenter, and noninferiority trial (INSPIRE) was performed.33 The total ischemic time was significantly shorter in the OCS group than in the SCS group (2.6 h versus 4.9 h for the first lung and 4.2 h versus 6.6 h for the second lung, P < 0.0001). The incidence of grade 3 PGD within 72 h after LTx was significantly lower in the OCS group (17.7% versus 29.7%, P = 0.015). The mean number of lung graft-related serious adverse events (including acute rejection, lung infection, bronchial anastomotic complications, and respiratory failure) within 30 d after LTx was 0.23 events per patient in the OCS group and 0.28 in the SCS group (noninferiority test P = 0.020). Then, a single-arm and multicenter pilot trial (EXPAND) was carried out by the same team to evaluate the efficacy of the OCS Lung device applied in ECD lungs.9 Eighty-seven percent (79/91) of ECD lungs were transplanted after OCS treatment, and a 99% survival rate at 30 d after LTx was achieved. Currently, 2 longer-term follow-ups of the INSPIRE and EXPAND trials are underway to evaluate the 5-y survival and the incidence of chronic lung allograft dysfunction (CLAD). Furthermore, Wallinder A and colleagues explored the incidence of CLAD and the mortality of recipients 4 y after LTx, which was similar (25% versus 32%, P = 0.42; 80% versus 70%, P = 0.31) in EVLP-treated lungs that were initially rejected and standard donor lungs.36 Other long-term results including graft function and quality of life also presented similar in ECD lungs treated with EVLP and conventional lungs after 5 y of follow-up.34

Providing Effective Rehabilitation

With regard to donor lungs with specific pathologies, such as pulmonary embolism (PE), infection, and edema, EVLP appears to be an excellent platform for rehabilitation. PE, a frequently detected condition that exists in donor lungs, is commonly considered a contraindication for LTx.37 However, Machuca TN and Inci I presented successful clinical LTx cases using lungs with PE that were subjected to urokinase therapy during EVLP, which was proven effective in treating patients without bleeding-related complications.32,38 Lungs with PE treated with fibrinolytic agents during procurement also exhibited markedly improved oxygenation when evaluated after EVLP. However, whether EVLP has inherent therapeutic effects remains inconclusive; nevertheless, the process is somewhat beneficial.39 Similarly, the addition of recombinant tissue plasminogen activator to the OCS lung perfusion circuit for donor lungs with massive pulmonary emboli was proven to be safe and efficient in LTx, followed by an uneventful postoperative course.40

EVLP has also been demonstrated to be an effective platform for rehabilitating infected donor lungs, in which adequate or supraclinical broad-spectrum antibiotics are administered without concerns about impairing other organs.41 After being treated with broad-spectrum antibiotics during EVLP, the infected human donor lungs showed reduced bacterial counts and endotoxin levels, along with improved compliance and oxygenation.42 Cytomegalovirus reactivation remains a significant cause of morbidity and mortality after LTx. The delivery of immunotoxin (F49A-FTP) during EVLP significantly attenuated the reactivation of cytomegalovirus in donor lungs.43 Moreover, germicidal light therapy for lungs infected by hepatitis C virus has been performed during EVLP in human lungs.44,45 Cypel M and colleagues conducted a single-center prospective trial to evaluate the safety and efficacy of hepatitis C virus-positive donor lungs subjected to EVLP plus ultraviolet C (UVC) irradiation in LTx.45 Although the recipient infection rates were not significantly different between the EVLP and EVLP plus UVC groups, there was a significant reduction in the viral load in the EVLP plus UVC group after LTx.

Additionally, donor lungs with neurogenic pulmonary edema, which is associated with brain death, are commonly discarded because of deteriorated oxygenation. However, Sanchez and colleagues reported the successful transplantation of a donor lung with neurogenic pulmonary edema, which was subjected to 3 h of EVLP rehabilitation.46 The lung was successfully salvaged and transplanted, with satisfactory posttransplant outcomes, including short periods of mechanical ventilation and hospitalization, as well as an unaffected quality of life.

Providing Reliable Diagnostic Assessment

Providing Physiological Variables

In addition to an effective therapy, EVLP has also been developed as an effective instrument for assessing and predicting the function of donor lungs in clinical LTx. During EVLP, the hemodynamic and ventilatory parameters of donor lungs can be observed on a monitor in real time. Furthermore, various physical methods, such as evaluation of the perfusate volume in the reservoir, X-ray examination, bronchoscopy, and lung ultrasound, can be instructive in assessing lung edema. Given the uncertainty in distinguishing parenchymal lung injuries, real-time computed tomography during EVLP has been proven feasible.47

However, it should be noted that the direct visual parameters may not adequately represent the quality of donor lungs, as these parameters remain normal for some time even when there is evident lung edema. Some physical parameters are to some extent predictive of donor lung function and are beneficial for decision-making regarding transplant suitability after EVLP. Donor lung weight appears to be an effective indicator of water gained and a predictor of transplant suitability. A higher donor lung weight after 2 h of EVLP has been reported to be significantly correlated with poor oxygenation and a lower transplant suitability rate after EVLP than a lower donor lung weight.48 It has also been found to be associated with a higher rate of grade 3 PGD and longer durations of ventilation and hospitalization.49 Additionally, the lung ultrasound score, determined using a binary method during EVLP, has shown better performance in discriminating declined donor lungs than objective parameters such as lung compliance.50 Electrical impedance tomography during EVLP has been introduced for assessing lung edema, with the values being inversely correlated with extravascular lung water, peak airway pressure, and lung injury score.51 Notably, the stress index, a ventilatory parameter derived from the pressure-time curve, demonstrated superiority over lung compliance in identifying injurious mechanical ventilation during EVLP, which was positively correlated with the duration of mechanical ventilation and the length of stay in the ICU and hospital.52 Therefore, these physical variables might be reasonable for predicting lung function before LTx or short-term posttransplant outcomes.

Detecting Chemical Biomarkers

In addition to physical variables, a variety of potential chemical biomarkers during EVLP have been identified to detect early detrimental signs, especially those underlying injuries that are not yet visible on physical inspection. Of note, inflammatory cytokines have been established as dominant contributors to lung injury, especially interleukin-8 (IL-8), which is strongly associated with the occurrence of PGD.53 The Toronto Lung Score (TLS2), calculated using a 2-plex cytokine index involving levels of interleukin-6 (IL-6) and IL-8 in the EVLP perfusate, precisely predicted the outcomes of EVLP with 87% accuracy and demonstrated an advantage over the canonical objective parameters.54 Similarly, the levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the EVLP perfusate were used to predict both the decision to transplant or decline following EVLP and the mid-term posttransplant outcomes.55 The receiver operating characteristic (ROC) curves of 1-y survival confirmed the prognostic signature, showing satisfactory prediction [area under the curve (AUC) of 0.93 for IL-1β and 0.95 for TNF-α]. More importantly, the level of IL-1β in the EVLP perfusate showed high accuracy in predicting mortality at 1 y posttransplant, with a diagnostic sensitivity of 83% and specificity of 100%.

Identifying early cellular death signals during EVLP may help to predict posttransplant outcomes. It is well known that danger-associated molecular patterns (DAMPs) are released during various types of cellular death. For further validation, Hashimoto investigated whether DAMPs in the EVLP perfusate could predict posttransplant PGD.56 Their results demonstrated that increased levels of high mobility group box 1 and M30 indicated a higher risk of donor lungs developing PGD. Similarly, the cell-free DNA in the EVLP perfusate, acting as another type of DAMP, has also been identified as a prognostic indicator of PGD, with area under the curve values of 0.718 and 0.733 for mitochondrial DNA and nDNA, respectively.57 Additionally, neutrophil extracellular traps could also be detected in the EVLP perfusate and were significantly associated with the duration of ventilation after LTx.58 Overall, EVLP has become an excellent platform for investigating early cellular processes occurring in donor lungs before LTx, and noninvasive perfusate samples can offer valuable predictive information about posttransplant outcomes.

Preclinical Evidence

As discussed above, EVLP has significantly increased the successful clinical use of ECD lungs. Importantly, EVLP creates a window between procurement and LTx, during which‚ function and viability of the donor lung can be assessed. EVLP not only is a feasible assessment tool in clinical LTx, but also allows numerous experimental studies for translational research. There have been promising preclinical results of various therapies using drug, gene, stem cell, and medical gas during EVLP. Based on compelling evidence, the following sections summarize the preclinical findings of EVLP in the optimization of donor lung function, providing potential therapeutic targets and strategies.

Providing Therapeutic Targets

Targeting Endothelial Protection

Based on the crucial role of endothelial integrity in maintaining the alveolar-capillary barrier, endothelial protection has attracted increasing attention in donor lung preservation.59,60 EVLP allows endothelial glycocalyx protection via the addition of a heparanase inhibitor, which resulted in better organ preservation and lung graft quality in rats.61 Similarly, because of the pivotal importance of circulating sphingosine-1-phosphate (S1P) in regulating endothelial integrity, the addition of S1P and its synthase sphingosine kinase 1 to the EVLP perfusate resulted in improved compliance and decreased pulmonary vascular permeability of donor lungs.62 Moreover, the endothelin axis activation surge is strongly associated with the pulmonary endothelial dysfunction induced by brain death, and the administration of an endothelin receptor antagonist during EVLP dramatically prevented physiological deterioration and improved the quality of lungs from brain-dead donor sheep.63 These data underscore the relevant role of endothelial protection during EVLP in improving lung performance.

Targeting Specific Receptors

Some studies have attempted to deliver agonists or antagonists of specific receptors during EVLP to improve donor lung function. β2-Adrenergic receptor, a G protein-coupled receptor, is widely distributed in smooth muscle and blood vessels. The infusion of its agonist during EVLP has been reported to have beneficial effects on glucose concentration, edema clearance, and dynamic compliance of donor lungs through a cyclic adenosine monophosphatedependent mechanism.64 Notably, high-dose inhalation of a β2-adrenoreceptor agonist during EVLP was associated with better oxygenation and increased tissue levels of cyclic adenosine monophosphate, which existed both at the end of EVLP and 4 h after LTx.65,66 Similarly, the adenosine A2A receptor and adenosine A2B receptor are also affiliated with the G protein–coupled receptor family. The potential of EVLP with targeted A2Areceptor agonist or A2Breceptor antagonist delivery to optimize lung function by downregulating inflammation has been highlighted.67,68 Taken together, these studies show that targeting specific receptors during EVLP can effectively improve donor lung function and attenuate ECD lung injury.

Targeting Mitochondrial Function

Because mitochondria are essential organelles for metabolism and cell function, the maintenance of mitochondrial function has emerged as a key target for organ protection in transplantation.69 However, mitochondria experience ischemia-related injury and become highly sensitive to ischemia-dependent events. Hence, mitochondrial dysfunction occurs during the SCS period, which may be related to ischemia-reperfusion (IR) injury and posttransplant PGD.70 Targeted delivery of protective agents for mitochondria during EVLP seems to be an effective therapeutic intervention. Delivery of the calcineurin inhibitor cyclosporin A into the perfusate in a rat model of EVLP resulted in attenuated inflammatory events, lower glucose consumption, and better posttransplant graft quality through regulation of the mitochondrial permeability transition pore.71 Similarly, ex vivo administration of trimetazidine, a metabolic modulator, also provides protection for donor lungs by inhibiting mitochondrial permeability transition pore, lipid peroxidation, and neutrophil activation.72 Moreover, the use of diazoxide during EVLP, which is a mitochondrial adenosine triphosphatesensitive potassium channel (mKATP) modulator, has been shown to improve lung physiological and metabolic parameters. During SCS preservation, the stopped blood flow triggered mechanosignaling, resulting in cell membrane depolarization and lowered intracellular potassium. The administration of diazoxide stimulated membrane hyperpolarization and could provide lung protection.73 These pieces of evidence present a strategy that may be used during EVLP for mitochondrial protection in donor lungs, avoiding the detrimental effects of SCS.

Targeting Inflammatory Pathways

Excessive inflammation, a major underlying mechanism of ECD lungs, is becoming a therapeutic target during EVLP. Ex vivo administration of various anti-inflammatory agents, such as steroids and heat shock protein-90 (HSP-90) inhibitors, has shown promising outcomes in dampening inflammation, especially the sterile immune response.74,75 Given the fundamental role of nuclear factor kappa B (NF-κB) in governing the release of proinflammatory cytokines, its inhibitor pyrrolidine dithiocarbamate (PDTC) was administered during EVLP, and lung edema, as well as the release of TNF-α and IL-6, was markedly decreased.76 Specifically, poly ADP-ribose polymerase 1 (PARP-1), known as a coactivator of NF-κB, was activated in donor lungs. Consistent with PDTC, delivery of the PARP-1 inhibitor 3-aminobenzamide (3-AB) into the perfusate of a rat EVLP model resulted in beneficial outcomes in damaged lungs, as indicated by reduced neutrophil infiltration and alleviated lung edema.77 Additionally, ex vivo treatment with the antioxidant N-acetylcysteine in the porcine lung following 24 h of cold ischemic storage alleviated the inflammatory response and improved posttransplant lung function via its antioxidant activity.78 Administration of alpha-1 antitrypsin (A1AT), a major acute-phase reactant antiprotease during acute inflammation, in the perfusate of a porcine EVLP model after 24 h of prolonged cold storage significantly reduced the release of interleukin-1α (IL-1α) and IL-8, as well as lung edema.79 In addition to these adverse effects of cold preservation, EVLP itself also generates a negative proinflammatory environment, which can be eliminated via leukocyte-depleting filtration, neutrophil elastase inhibition, triptolide administration, and cytokine adsorption.80-83 Thus, the clinical application of these anti-inflammatory strategies during EVLP is eagerly awaited.

Targeting Immunogenicity

Acute rejection induced by uncontrolled alloimmunity remains a serious and elusive problem following LTx. EVLP appears to provide an opportunity to modulate the immunogenicity of donor lungs before transplantation. Because of the high immunomodulatory ability of regulatory T cells, Miyamoto E and colleagues reported positive immunomodulatory effects using expanded recipient-derived regulatory T cells in the lungs of rats during EVLP, followed by successful LTx without adverse effects.84 Depleting passenger leukocyte therapy during EVLP in a porcine lung model also exhibited inhibitory effects on acute rejection by preventing donor leukocytes from migrating to recipient lymph nodes and reducing recipient T cell infiltration into the donor lung.85 Moreover, in the EVLP porcine model, perfusion with human blood modified by the addition of polymer GAS914, a soluble trisaccharide-polylysine conjugate, showed great potential in binding xenoreactive human antibodies and preserving the function of a porcine lung xenograft, thereby preventing hyperacute rejection in lung xenotransplantation.86 These studies highlight the potential of donor lung immunomodulation via EVLP before transplantation in decreasing posttransplant rejection.

Allowing for Therapeutic Strategies

Protective Gas Inhalation

Given that donor lungs are mechanically ventilated during EVLP, several experimental studies using animal lungs have explored the potential of protective gas inhalation, which involves hydrogen sulfide, carbon monoxide, sevoflurane, hydrogen, and nitric oxide (NO), in improving donor lung quality and posttransplant outcomes. Protective gases have been reported to exert positive effects on donor lungs via various mechanisms, including mechanisms inhibiting inflammation, oxidation, and apoptosis, as well as via metabolic regulation.87-90 Notably, antibacterial effects of continuous NO inhalation in porcine lungs during EVLP have also been observed, without the accumulation of methemoglobin and toxic nitrogenous compounds, which indicates the potential of EVLP for use in the treatment of infectious donor lungs using high-dose NO.91 Although these gases have not yet been used clinically, this approach seems worthy of consideration in future clinical practice.

Lung Lavage and Surfactant Replacement

EVLP has provided an excellent platform for the delivery of surfactant and lavage therapy for donor lungs suffering from gastric acid aspiration, which is a common condition of brain-dead donors. Inci and colleagues investigated the effects of surfactant therapy on acid-damaged porcine lungs during EVLP, which improved graft function with lower PA pressure, higher oxygenation, and decreased inflammation compared with the control group.92-94 Nakajima D described an optimized strategy through a combination of surfactant replacement and lavage therapy during EVLP in porcine lungs, which not only resulted in improved lung function during EVLP but also resulted in superior lung function after LTx.95 In clinical settings, serious gastric-induced damage often renders donor lungs unsalvageable for clinical LTx. This therapy has practical limitations and is awaiting validation in clinical EVLP.

Gene Therapy

Over the last decade, researchers from Toronto have focused efforts on gene therapy during EVLP, obtaining satisfactory results. IL-10, an anti-inflammatory cytokine carried by an adenoviral vector, was delivered to the segmental bronchus using a bronchoscope during EVLP.96 Beneficial effects were achieved after EVLP in porcine lungs, as indicated by improved oxygenation capacity and reduced cytokine levels.96 Moreover, ex vivo delivery of the IL-10 gene turned out to be superior to in vivo delivery because vector-related systemic infection and immunosuppression were avoided.97 Then, they further evaluated the effect of IL-10 gene delivery on posttransplant lung function in a porcine EVLP model. The results showed improved lung performance and reduced tissue inflammation 7 d after LTx, as well as an attenuated allograft rejection response.98 However, because of the toxicity of the adenoviral vector, future studies should consider nonvector gene delivery strategies, if feasible.

Cell-based Therapy

Promisingly, cell-based therapy during EVLP has shown potential for use in donor lung reconditioning.99 Unlike airway-based gene delivery, the intravascular delivery of mesenchymal stem cells (MSCs) to porcine lungs during EVLP has been proven to be more effective than delivery via the intrabronchial route, resulting in prolonged MSC retention and decreased IL-8 levels, as well as increased growth factor levels because of the paracrine capacity of MSCs.100,101 Another similar resource for cell-based therapy during EVLP is multipotent adult progenitor cells (MAPCs), which have a great proliferation capacity and low senescence rate. MAPCs have been reported to exert significant anti-inflammatory effects on porcine lungs during EVLP.102 More importantly, stem cell-derived extracellular vesicles (EVs) are emerging as a promising treatment for lung injury without concerns about cell-related immunogenicity or malignant transformation. Administration of EVs to the lung in a rat EVLP model led to improved lung metabolism and tissue integrity.103 These data demonstrate that stem cells and their derived EVs have broad prospects for application during EVLP for donor lung recovery.

Cross-circulation EVLP With Xenogeneic Support

Xenogeneic cross-circulation EVLP is an optimized technique in which the blood is continuously exchanged between EVLP-treated lungs and living swine. The combination of EVLP and xenogeneic transplant immunology has provided an excellent platform enabling normal physiological support for donor lung recovery.104,105 Surprisingly, indications of acute immunological rejection were not detected with the administration of immunosuppressive drugs and recombinant cobra venom factor. Better lung performance was achieved after 24 h of cross-circulation EVLP between a human donor lung and a swine host, showing an improvement in gas exchange capacity and dynamic compliance. The airway seemed to be recovered without evidence of airway secretions, although mild secretions were observed before EVLP. Additionally, levels of inflammatory cytokines in the serum and the bronchial lavage fluid were decreased, indicating that cross-circulation EVLP with xenogeneic support is a promising therapy for the recovery of donor lungs.106

CURRENT PROBLEMS AND LIMITATIONS OF EVLP

When evaluating the overall value of EVLP, although tremendous achievements have been made, some existing limitations should be considered. It is worth noting that the EVLP process per se is equivalent to another form of IR, and inflammatory responses and oxidative stress occur when donor lungs are reperfused after SCS. Coupled with external exposure to circuit materials, exposure to a harmful proinflammatory environment is thus inevitable for donor lungs. Although the ventilation and circulation of donor lungs are restored during EVLP, as is normothermia, the environment is not entirely based on physiological conditions because of the lack of a hepatic or renal clearance mechanism and the stimuli from recipient blood cells or proteins. Furthermore, despite the efficiency, lung edema following EVLP or LTx is sometimes insurmountable, which has remained an intractable problem troubling transplant surgeons and perfusionists. Hence, recognizing the limitations of EVLP and optimizing the corresponding therapeutic measures will be beneficial for the popularization of EVLP in LTx, as discussed below.

Endogenous Capacity to Produce a Proinflammatory Environment

The proinflammatory effect of EVLP on donor lungs may presumably be associated with the interactions between the artificial circuit components and the perfusate constituents, as well as IR-derived inflammation. However, this proinflammatory effect is not significantly affected by donor lung characteristics.14 There are also no indications of histopathological deterioration of donor lungs even though increased inflammatory cytokine levels may be detected during EVLP, which may be associated with the closed-loop signature of EVLP, without the hazards of continuous inflammatory cell recruitment from the blood.107 In EVLP-treated human donor lungs, Elgharably et al showed that the production of inflammatory cytokines increased over time in the EVLP perfusate compared to the baseline levels.108 The expression of microribonucleic acid (miR), especially miR-17 and miR-548b, was upregulated during EVLP, which was related to macrophage migration and the inflammatory response. Baciu and colleagues focused on the discrepancy between EVLP lungs and non-EVLP lungs at a transcriptomic level.109,110 Their results demonstrated that the TNF receptor-1/2 signaling pathways and macrophage migration inhibitory factorrelated pathways were enriched in EVLP lungs.109 EVLP induced a marked upregulation of inflammation-related gene expression. Notably, innate immune signaling was involved in the greatest number of pathways and was considered a major molecular event during EVLP.110 Similarly, another transcriptomics investigation showed that the expression of innate immune signaling, HSP and macrophage activation-related genes was induced in EVLP lungs.111 These data suggest that targeting these pathways during EVLP may allow for successful lung reconditioning before transplantation.

Induction of Metabolic Changes

Donor lungs also undergo a marked metabolic change during EVLP. Because of the absence of gluconeogenesis and limited glycogen storage, donor lungs primarily depend on glycolysis to meet their metabolic demands. However, adverse consumption of glucose, the major energy substrate, during EVLP was detected in donor lungs, and a strong positive correlation between glucose consumption and lung edema was demonstrated, which highlights the need to add fresh perfusate over the course of EVLP.112 Using metabolomics, Shin and colleagues analyzed the metabolic behavior of human donor lungs subjected to EVLP.15 Not only the consumption of carbohydrate energy substrates but also the accumulation of metabolites (amino acids and nucleic acids) and metabolic byproducts, especially lactate, were emphasized in their studies.15 Using microdialysis, Mazzeo and colleagues identified increased levels of glutamate, pyruvate, glucose, and lactate in the EVLP perfusate and interstitial fluid as potential metabolic markers of lung damage.113 Additionally, EVLP induces alterations in L-arginine metabolism, resulting in an imbalance between L-arginine and NO synthases and reduced NO production.114 Therefore, it is possible that EVLP stimulates a process of metabolic reawakening, indicating the need for further investigations to facilitate homeostasis of the donor lung. Targeting the development of new perfusates with appropriate nutrients and clearance equipment seems to be a promising direction of future research.

Lack of a Clearance Mechanism

Additionally, there is a gradual buildup of metabolites; however, the corresponding clearance mechanisms, such as the kidneys and liver, present under normal physiological conditions are absent in the EVLP system, which is not favorable for the reconditioning of donor lungs and may be detrimental to posttransplant outcomes. Based on the Toronto protocol, Takahashi et al modified the perfusion method using a syringe infusion pump for continuous delivery of Steen and a multichannel syringe pump for continuous collection of the perfusate, resulting in a significant reduction in IL-6 and IL-8 levels in EVLP perfusate.115 Nevertheless, because of a greater demand for Steen, this modified perfusion method is of operational complexity and high cost. Wei et al demonstrated the safety and feasibility of installing a dialyzer in the EVLP circuit of porcine lungs instead of frequently exchanging the perfusate. Their results not only suggested an extended time of 12 h for EVLP operation but also improved donor lungs homeostasis.116 Takahashi et al explored the effect of dialysis on cytokine and gene expression profiles in porcine lungs during EVLP in a continuous venovenous hemofiltration manner. As a result, continuous dialysis was proven to maintain metabolic homeostasis with no impact on gene expression profiles or donor lung functions.117 Hence, it is possible to conclude that the continuous dialysis protocol is feasible during EVLP.

Lack of Bronchial Arterial Circulation

It is essential to realize that the recovery of bronchial arterial circulation (BAC) is not routinely achieved in the current LTx field because it consumes substantial effort and prolongs the ischemic time, thereby counteracting the transplantation-derived benefits. Moreover, bronchial circulation is absent from the time of the initial procurement, resulting in disruption of the pulmonary microvasculature, and appears to correlate with the development of CLAD, including bronchiolitis obliterans syndrome.118 To maintain BAC during donor lung preservation, Tanaka et al developed a dual-perfusion EVLP circuit with concurrent BAC and PA circulation perfusion, which yielded better lung performance after EVLP compared to the standard EVLP; the results indicated improved metabolism and decreased inflammation, as well as satisfactory posttransplant outcomes with increased microvasculature and improved lung function.119 Similarly, Tane S et al optimized the perfusion protocol for donor lungs during procurement via a BCA sparing approach; subsequent evaluation of the lungs by EVLP demonstrated preserved microvasculature and improved lung function in groups treated with the BAC sparing approach.120 These modified strategies highlight the importance of BAC maintenance during lung preservation and EVLP before LTx.

Generation of Ventilator-induced Lung Injury

Although providing ventilation for donor lungs, EVLP also inevitably generates ventilator-induced lung injury (VILI). Commonly, current EVLP devices utilize the positive-pressure ventilation (PPV) protocol, which is more likely to induce ventilatory heterogeneity throughout the lung parenchyma and is highly correlated with VILI.121 Given that negative-pressure ventilation (NPV) is a more physiological ventilatory strategy than PPV, several studies have explored whether NPV could be beneficial for better lung performance during EVLP in preclinical models.122–124 Consequently, NPV demonstrated superiority over PPV, as indicated by reduced inflammatory cytokines and mitigated lung edema in the treated lungs.122,123 Of note, the NPV protocol during EVLP has also been proven to be effective and feasible in a single-arm interventional clinical trial involving 12 ECD lungs, in which the early posttransplant outcomes were comparable to those of standard criteria donor lungs.124 Additionally, researchers have attempted to investigate the effects of prone positioning on donor lungs during EVLP, which is a well-established intervention for patients subjected to acute respiratory distress syndrome (ARDS).125 Prone positioning resulted in better performance of donor lungs during EVLP, which was mainly associated with a more homogeneous distribution of interstitial fluid.126-128 In addition to changes in the pressure and position of ventilation, advantages of other optimized ventilatory patterns during EVLP, including personalized positive end-expiratory pressure titration, pressure-directed airway pressure release, and marathoners’ breathing patterns, have also been suggested.129-131 Nevertheless, most studies have been based on preclinical models, and more clinical trials are required to validate the above interventions.

Economic Considerations

Currently, the high cost of EVLP is another dilemma in LTx. A multicenter observational study evaluated the cost effectiveness of EVLP in LTx and showed that the cost of EVLP was far higher than that of the standard process, despite an increase in the number of eligible donor lungs for transplantation.35 Notably, in addition to the costly equipment and single-use consumables, the cost of increasingly frequent Steen replacement needs to be taken into consideration. Increases in the usage of extracorporeal membrane oxygenation and in the ICU length of stay when EVLP is applied in ECD lungs are also important considerations. Furthermore, in pursuit of maximum cost effectiveness, the rate of conversion to LTx of ECD lungs subjected to EVLP should be increased via various rescue interventions. Ultimate targets of the best posttransplant outcomes for recipients and the lowest healthcare costs for EVLP are of paramount importance for the broader adoption of EVLP in LTx.

CONCLUSION AND FUTURE PERSPECTIVES

EVLP undoubtedly represents the most revolutionary technology practiced in the landscape of LTx over the past few decades and has achieved a remarkable reputation for improving the quality of ECD lungs and expanding the lung donor pool. By summarizing relevant works in the literature, our review has provided a deeper understanding of the advantages and disadvantages of EVLP (Figure 2). Also, a detailed description of the evolution and protocols of EVLP, along with numerous lung-protective agents and therapeutic measures that can be applied during EVLP, could provide the opportunity for more perfusion physicians, surgeons, and anesthesiologists to be exposed to EVLP.

F2
FIGURE 2.:
Advantages and disadvantages of EVLP. Accumulating evidence has confirmed the advantage of EVLP for evaluating and optimizing donor lung function, as well as expanding the donor pool. However, EVLP is not a technological panacea, and not all donor lungs are salvageable. The potentially adverse inherent effects of EVLP have been summarized. Recognizing both the advantages and disadvantages of EVLP is of great importance in promoting its broader clinical usage. More importantly, the evolution toward an ideal EVLP protocol calls for more effective approaches in the future. BAC, bronchial arterial circulation; EVLP, ex vivo lung perfusion; LTx, lung transplantation; VILI, ventilator-induced lung injury.

A new era has dawned for EVLP in LTx; however, the potential limitations associated with future directions of EVLP need to be considered. First, most clinical studies have been designed to demonstrate the safety and efficiency of EVLP; however, there have been very few clinical conclusions associated with failed EVLP thus far. Additionally, clinical conclusions associated with the superiority of EVLP regarding long-term recipient outcomes such as CLAD and survival are currently limited, which underscores the need for more prospective, randomized control trials. Second, most experimental results are limited and considered preclinical evidence; more therapeutic strategies and diagnostic approaches are expected to be verified clinically to further enhance donor lung utilization in LTx. Third, as EVLP does not operate under entirely physiological conditions, it must be recognized that when inappropriately used, it has the potential to induce edema and other deleterious effects. Finally, portable EVLP would facilitate donor lung procurement and mitigate the hazards elicited of cold ischemic storage. With further investigation, EVLP should be developed as an ideal new platform with the capacity for long-term and portable operation. Therefore, future research should attempt to eliminate the factors that have limited the widespread usage of EVLP. Collectively, as the substantial benefits and potential limitations of EVLP in LTx continue to be elucidated, the possibility of EVLP in LTx becoming a routine practice increases. The results of a number of ongoing experimental and clinical trials on EVLP are eagerly awaited.

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