Although over 120, 000 patients are currently on the waitlist for a lifesaving or life-extending organ in the United States, only 36, 529 transplants were performed in 2018.1 The shortage of available donor organs has pushed for the usage of marginal donor organs. However, the discard rate of organs remains high because of old age, previous organ damage, or predisposing condition and anticipated preservation injury. In addition, the usage of marginal donor grafts is also associated with a higher risk of developing posttransplant complications.2,3 Many of these complications are exacerbated by severe ischemia-reperfusion injury (IRI),4,5 which occurs when organs are preserved in a hypoxic static cold storage (SCS) environment, the traditional method of organ preservation. Therefore, organ machine perfusion (MP) has emerged as a promising alternative in kidney, liver, lung, heart, and pancreas preservation.5
Compared with SCS, MP allows for organs to be continuously perfused with oxygen and nutrients during the preservation period, thus limiting the period of cold ischemia. MP in most solid organ transplantation can be divided into normothermic MP (NMP; 35.5–37.5°C) and hypothermic MP (HMP; 1–8°C), and subnormothermic MP (20–35.5°C) has also been explored in some studies.6 Clinical trials involving NMP have shown promising results in lung, liver, and kidney transplantation. Keeping organs in a normothermic setting decreases the length of cold ischemia time and allows for oxygen and nutrients to be delivered to the organ. In addition, NMP allows easier organ assessment during the perfusion period. Clinical trials in HMP have been explored mostly in kidney transplantation, with some trials in liver transplantation as well. Compared with NMP, HMP is less complex and requires lower cost to implement, because it does not require oxygen carriers and all of the supplements necessary to keep optimal organ metabolism. In addition, HMP allows for some variables of organ assessment to be measured, such as flow rate and mitochondria oxidative function metabolites.7,8
Both NMP and HMP can act as platforms for injured organs to be repaired by the addition of therapeutic agents in the perfusate.9 Numerous MP therapeutic agents (MPTAs) have been tested in animal and human models, such as antiinflammatory agents, vasodilators, antiinfection agents, mesenchymal stem cells (MSCs), gene therapy agents (siRNA and shRNA), and defatting agents (Figure 1). This comprehensive review will examine and summarize all of the therapeutic agents used in MP of lung, liver, kidneys, and heart. Although MP has also been studied in pancreas10,11 transplantation, no MPTA studies have been published to date. Here, we specifically define MPTA as an agent that is added to the machine perfusate during ex situ MP in addition to standard perfusate (preservation solution) with the intention to modulate the graft. We are not considering here as MPTA insulin, heparin, glucose, basic nutrients, and oxygen.
LUNG MACHINE PERFUSION THERAPEUTICS
Lung transplantation is the most effective treatment for end-stage lung disease.12 Despite the benefits of this lifesaving procedure, only 22.6% of the recovered lungs were used for transplantation in a 2017 report.13 This high discard rate can be partly attributed to the strict inclusion criteria for acceptable donor lungs. Lung transplantation recipients suffer from a high rate of primary graft failure due to IRI.14 Marginal lung grafts stored under traditional SCS are especially susceptible to IRI due to prolonged cold ischemia time.14 Recently, numerous clinical trials have demonstrated that marginal lungs transplanted after normothermic ex situ lung perfusion (ESLuP) have similar posttransplant outcomes compared with higher quality lungs preserved by SCS.15-17 A wide array of MPTAs have been investigated under NMP to further prevent the damage caused by IRI, some of which have even resulted in successful transplantation of would-be rejected lungs. These agents are summarized in Tables 1–3.
Mesenchymal Stem Cell and Related Therapies
MSCs are bone marrow–derived multipotent cells that secrete paracrine soluble factors with numerous therapeutic effects. The therapeutic potentials of MSCs have been explored in numerous diseases, such as myocardial infarction, sepsis, and diabetes. MSCs have also been shown in several in vivo lung models to ameliorate acute lung injury through modulation of the inflammatory response.52,53 Several laboratories have since then investigated the protective effects of adding MSCs into machine perfusate during lung preservation. MSC, MSC-derived microvesicles, and multipotent adult progenitor cells (another bone marrow–derived stem cell) have all been studies under ESLuP; these agents are summarized in Table 1.
Lee et al19 first demonstrated the potential of using MSCs as an MPTA in 2009. In an ESLuP model, this laboratory treated–rejected human donor lungs injured by Escherichia coli endotoxin. The intrapulmonary administration of allogenic human MSCs improved alveolar fluid clearance (AFC), lung endothelial permeability, and pulmonary edema compared with ESLuP alone. Antiinflammatory effects were also demonstrated through a nonsignificant decrease in neutrophil cell count. In addition, the laboratory was able to show that the MSCs’ antiinflammatory effects were largely due to the secretion of keratinocyte growth factor, as silencing of this growth factor reduced the protective effects of MSC by nearly 80%.19
In a follow-up project, Lee et al20 used the same model to test donor lungs that were injured by live E. coli bacteria. Once again, improvements in AFC and antiinflammatory effects were observed in treated lungs. More interestingly, the MSCs also lowered the alveolar bacterial load in a dose-dependent manner and increased alveolar macrophage phagocytosis activity, thus demonstrating the antimicrobial effects of MSCs. McAuley et al,21 as a follow-up study, tested the therapeutic effects of MSCs using rejected donor lungs not injured by bacteria. Compared with ESLuP alone, MSC-treated lungs had significantly improved AFC to a normal level after 4 h of perfusion.
Mordant et al18 investigated the optimal delivery route and dosage of MSCs using an ESLuP model with porcine lungs. They concluded that the intravenous delivery of MSC at a dosage of 150 × 106 cells led to the most optimal decrease in interleukin-8 (IL-8), a proinflammatory cytokine. This dosage is nearly 30 times higher than that of the studies done by Lee et al and McAuley et al, who were able to show physiological improvements at a much lower dosage. More studies using the human lung models are needed to examine the true optimal dosage of MSCs in ESLuP.
Microvesicles derived from MSCs have also been investigated as a potential MPTA. MSC-derived microvesicles are circular fragments of membrane that contain biologically active materials, including mRNAs. In vivo models have shown that these microvesicles have similar therapeutic properties as MSCs.54 Additionally, the microvesicles have a lesser risk of causing tumor formation compared with MSCs. In a murine ESLuP model, Stone et al22 investigated the immunomodulatory mechanisms of MSC-derived microvesicles. A significant decrease in proinflammatory cytokines (IL-7, tumor necrosis factor alpha [TNF-α], CXCL1, and High Mobility Group Box 1) and a significant increase in antiinflammatory agents (keratinocyte growth factor, IL-10, and prostaglandin E2) were observed in the treated mice.
The therapeutic potentials of MSC-derived microvesicles have also been tested in 2 human lung models. Gennai et al23 used rejected human donor lungs to show that the administration of microvesicles significantly improved AFC and lung compliance. In a related study, Park et al24 administered microvesicles in human lungs that were damaged by E. coli, and these treated lungs had significantly lower bacterial load, in addition to superior AFC and endothelial permeability, compared with control.24
Multipotent adult progenitor cells are another type of bone marrow–derived stem cell type that has generated interest in the field of MP. Two ESLuP studies have shown that these cells can significantly reduce inflammatory markers in human26 and porcine25 models. However, no significant physiological differences were observed compared with ESLuP alone.
Lung Lavage and Surfactant Replacement
Gastric acid aspiration is a process that can lead to damage of the alveolar and capillary networks of the lung, and in turn, acute lung injury and aspiration pneumonitis.55 This acidic damage causes inflammation and surfactant dysfunction, both of which can contribute to primary graft dysfunction.56 Therefore, lungs damaged by aspiration is a common reason for organ rejection. Several laboratries have examined the potential of performing bronchoalveolar lavage, followed by exogeneous surfactant administration, during ESLuP to recover lungs injured by aspiration. Two types of exogenous bovine extract surfactants were used in these studies, both of which contained mainly surfactant proteins B and C (Curosurf and bovine lung extract surfactant).
Inci et al35 used a porcine ESLuP model with lungs injured by hydrochloric acid and pepsin to mimic gastric aspiration. Lungs that were immediately treated by lavage and surfactant had superior pulmonary vascular resistance (PVR), better oxygenation, and lower pulmonary edema. However, no significant differences were noted in the levels of inflammatory cytokines.
In the clinical setting, aspiration damage is typically diagnosed via radiology and histology many hours after its onset. To account for this, Khalifé-Hocquemiller et al37 treated aspiration-injured lungs after 24 h of gastric juice-induced injury. Once again, treated lungs demonstrated superior PVR and oxygenation. In addition, significantly lower levels of IL-6 were also noted in the treated lungs;37 this additional finding demonstrates lavage and surfactant administration have a more prominent protective effect in lungs damaged by aspiration followed by warm ischemia.
The above results were further corroborated by 2 porcine transplantation models. Porcine lungs were damaged by aspiration injury followed by 436 and 638 h of ESLuP, and these lungs were then transplanted into recipient pigs and reperfused for 4 h. Lavage and surfactant administration were performed immediately before ESLuP. In both studies, treated lungs demonstrated superior oxygenation, higher compliance, and lower IL-1β and IL-6 levels in the blood. Nakajima et al38 specifically tested the minimal surface tension of isolated surfactant using a biophysical functional analysis and demonstrated that surfactant from treated lungs had significantly lower surface tension. In summary, exogenous surfactant, administered after bronchoalveolar lavage, is a promising MPTA that can be used to treat lungs damaged by gastric acid aspiration. Future studies involving human lung models are needed to confirm its efficacy.
Adenosine Receptor Agonist and Antagonists
Adenosine is naturally released by the body under conditions of cellular stress and mediates proinflammatory and antiinflammatory effects depending on the effector tissue and receptor type.57 The 4 known adenosine receptors are A1R, A2AR, A2BR, and A3R, all of which are expressed on human lungs. Prior studies using animal models have already demonstrated A1R, A2AR, and A3R agonists’ role in attenuating IRI.58-60 Therefore, adenosine is one of the 2 major categories of small compounds being investigated as a potential MPTA, the other being β-adrenergic agonists (Table 2).
Emaminia et al28 first examined the potential of using adenosine receptor agonists and antagonists as potential MPTAs during ESLuP. In a porcine model, retrieved lungs that were under 14 h of cold storage were treated with an A2AR agonist. Superior oxygenation, lower pulmonary edema, and inflammation were observed compared with lungs treated by ESLuP alone. In a subsequent study, Stone et al27 used a murine ESLuP model to examine specific gene expression profile changes that occur when A2AR agonists are added to the perfusate. They noticed a significant decrease in numerous inflammatory pathways, which led to reduced pulmonary edema, lower inflammatory cytokine levels, and superior lung function.27 Finally, in a porcine transplantation study, Huerter et al30 showed that the administration of A2AR agonists during ESLuP can improve oxygenation after transplantation.
In addition to A2AR agonists, 2 studies also investigated protective effects of using A2BR antagonists as a potential MPTA. A2BR has both proinflammatory and antiinflammatory effects, and its overall effect on lung injury depends on several factors, such as injury condition and type cells involved. Using murine and porcine ESLuP models, Huerter et al30 and Charles et al31 demonstrated that A2BR antagonists can alleviate damage caused by warm ischemia. The treated animals had superior lung compliance3031 and decreased inflammatory markers.31
Two types of β-adrenergic agonists have been tested as MPTAs in porcine and canine models. Salbutamol regulates fluid transport using sodium-dependent mechanisms and may be used as an MPTA to lower pulmonary edema during ESLuP. Valenza et al32 showed that salbutamol infusion during ESLuP lowers the level of glucose in the perfusate. The concentration of glucose, shown in a prior study, is directly related to the degree of pulmonary edema.61 Furthermore, salbutamol also demonstrated vasodilatory effects that may improve oxygenation during ESLuP; treated lungs demonstrated lower PVR and compliance compared with control.32
Inhaled procaterol has also been shown to improve AFC through a cAMP-dependent mechanism.62 Transplantation models have previously shown that procaterol administration before procurement can alleviate warm-ischemic injury.6364 Therefore, Kondo et al33 investigated whether procaterol inhalation during ESLuP can have similar protective effects in a canine model. Treated lungs had improved arterial and airway pressure, in addition to lower PVR and higher lung compliance. In addition, total adenosine nucleotide levels were also observed after treatment, reflecting superior energy storage.33 Hijiya et al34 corroborated these results in a canine transplantation model, showing that transplanted lungs that were treated with procaterol had superior pulmonary functions and superior energy storage.
The postmortem microthrombi formation is another major cause for lung dysfunction and organ rejection, especially for donation after circulatory death (DCD) lungs. Fibrinolytic agents, such as urokinase, have been shown to have protective effects when infused into the donor lungs after cardiac arrest,6566 which lead to the possibility of using fibrinolytic agents as an MPTA. Inci et al39 used a porcine ESLuP model to show that urokinase, when added to the perfusate of ESLuP, can lower PVR, improve oxygenation, and lower pulmonary edema. The same group later used urokinase to treat a pair of human donor lungs that were damaged by pulmonary embolus. After 3 h of ESLuP with urokinase, significant improvements in compliance and oxygenation were observed, and the lungs were eventually transplanted without complications.40 In a similar manner, Machuca et al67 used a different thrombolytic agent, alteplase, to resuscitate a pair of human donor lungs that were rejected because of pulmonary embolism. Increased thrombolytic activity and decreased pulmonary arterial pressure were observed after 6 h of ESLuP, and the lungs were eventually transplanted without complications.
Drugs Targeting Microorganisms
The donor-to-host transmission of infectious microorganisms is a well-known threat to the immunosuppressed recipient, and ESLuP can act as a platform for treating lungs that are infected by bacteria and fungi. Andreasson et al42 first demonstrated this possibility by treating 18 marginal donor lungs with a broad-spectrum antibacterial agent, meropenem. After the discovery of fungi load in the first 3 pairs of lungs, an antifungal agent, amphotericin B, was also added to the perfusate. Significant reduction in bronchoalveolar bacterial and fungi load was observed in the treated lungs, and 4 infected lungs were able to be cured and transplanted without complications. Nakajima et al43 later investigated the possibility of using multiple antibacterial agents to treat rejected lungs with polymicrobial infections. In addition to a significant decrease in bacterial load in the bronchoalveolar lavage, numerous inflammatory markers, such as TNF-α and IL-1β, were also significantly reduced. In turn, the treated lungs also demonstrated superior compliance and oxygenation compared with control.43
A recent study demonstrated the potential of using light therapy as an MPTA to inactivate hepatitis C virus (HCV) in donor lungs using a modified ESLuP device. Up to 20% of lung donors in the United States are tested positive for HCV,68 and many of these donors are victims of overdose death—which means they have young and relatively healthy lungs. In a modified ESLuP model with an illumination device, Galasso et al44 used photodynamic therapy using methylene blue activated by red light irradiation to decrease HCV RNA levels by 98% in the perfusate and by 91% in lung tissue. UV C irradiation was also administered to a separate group of lungs and demonstrated similar virucidal effects.44 The results of this study have led to a prospective pilot clinical trial involving the transplantation of 22 HCV-positive lungs.69 Half of these lungs were treated with UV C irradiation, whereas the other half underwent ESLuP alone. Lungs treated with radiation demonstrated significantly lower recipient viral loads in blood within the first week after transplantation.69
The release of proinflammatory cytokines is a major cause of donor lung damage and rejection. A Toronto Group has conducted several studies using human and porcine models to investigate the possibility of using interleukin-10 (IL-10) gene therapy to suppress lung inflammation during ESLuP. Unlike in vivo gene therapy, in which gene delivery must be administered systemically, gene therapy during ESLuP allows for a more isolated delivery with less dosage and fewer systemic side effects. Cypel et al45 treated 10 discarded human donor lungs using adenoviral vector encoding human IL-10 (AdhIL-10) for 12 h. Treated lungs had superior oxygenation and lower PVR compared with control. In addition, a shift from proinflammatory to antiinflammatory cytokine production was observed.45 Yeung et al46 then used a porcine transplantation model to show that ex situ delivery of AdhIL-10 led to superior posttransplant lung function and less vector-related inflammation compared with in vivo delivery—demonstrating that ESLuP is a superior gene transduction platform. As a follow-up study, Machuca et al41 used a similar porcine model with a 7-d posttransplantation period to examine the long-term effects of using AdhIL-10 therapy. Treated lungs had superior posttransplant lung function and less inflammation, and interferon-γ suppression was demonstrated up to day 7.
Gene Modulating Agents
The complex process of IRI activates numerous genes that lead to cell apoptosis and tissue injury.4 Therefore, gene-silencing agents such as siRNA and shRNA have been explored in solid organ transplantation to reduce graft injury. In lung transplantation, in particular, the intratracheal delivery of siRNA and shRNA has been shown to alleviate IRI in in vivo murine models. These studies targeted proapoptotic and proinflammatory proteins, such as Fas, caspase, myeloid differentiation protein-2, and p38 mitogen-activated protein kinase.70 Although these results demonstrate the potential of RNA interference in lung transplantation, the in vivo intratracheal delivery of these agents is limited in the clinical setting, because it requires an invasive setup and requires several hours of intubation. ESLuP is a much more practical method to deliver RNA agents in an ex vivo and more targeted setting.
The ESLuP delivery of shRNAs using lentiviral vectors has already been explored to silence major histocompatibility complex antigens to avoid acute cellular rejection of donor lungs posttransplantation. Figueiredo et al47 showed that 2 h of ESLuP treatment with shRNAs targeting swine leukocyte antigens led to over 50% silencing of these endothelial antigens. No significant adverse effects were observed by the administration of lentivirus. This ESLuP method of delivering shRNAs can be applied to target antiapoptotic and antiinflammatory agents, such as those used in the aforementioned in vivo studies.
Other Therapeutic Agents
Numerous other MPTAs have been tested using murine and porcine models, but many of these results have yet been replicated using a transplantation or human lung donor models, and these are listed in Table 3. Briefly, several antiinflammatory agents, such as methylprednisolone,49 α1-antitrypsin,48 and neutrophil elastase inhibitors51 have been shown to improve pulmonary physiology and lower inflammatory markers in ESLuP porcine models. Hydrogen gas, a potent free-radical scavenger, has been used in ESLuP to improve pulmonary function and lower inflammation.71 Sphingo-1-phosphate, a regulator of endothelial barrier, has been shown to decrease endothelial vascular permeability.50 Although many of these agents will need to be further evaluated using transplantation and human lung models, they are promising therapeutic options in ESLuP.
Liver Machine Perfusion Therapeutics
Liver preservation using MP is also an evolving field with numerous recent and ongoing clinical trials demonstrating its superior performance compared with SCS.72-76 Since the growth of new waitlist registrants continues to exceed the number of available donors,77 the usage of marginal livers (steatotic, elderly, and DCD livers) has been increasing. The benefits of ex situ liver machine perfusion (ESLiP) are especially apparent in the context of marginal liver transplants because ESLiP offers a platform for the reconditioning of these livers before transplant. In addition, numerous biomarkers in the perfusate and in bile, such as bilirubin, aspartate aminotransferase, alanine aminotransferase, and lactate, have been shown to be associated IRI and hepatobiliary injury.8,78-85
Several laboratries have already examined the potential of using MPTA during ESLiP, and these studies can be summarized in Table 4. Almost all studies were conducted under normothermic settings. Two studies were conducted under subnormothermic settings, and 1 study was under hypothermic setting. Two major categories of liver MPTA are defatting cocktails86-88 and vasodilators,89-92 both of which have been tested in multiple rat and porcine ESLiP and transplantation models. Additional studies involving human livers and clinical trials will need to be conducted to solidify these promising results.
Steatotic livers account for a large portion of organ rejection because their usage is associated with a higher rate of primary graft dysfunction.100 Therefore, several labs have examined the possibility of reconditioning steatotic livers using ESLiP. Jamieson et al86 have shown that NMP alone can significantly reduce steatosis in a porcine ESLiP model. In this study, porcine liver steatosis was induced using streptozocin (a drug that causes hyperglycemia) and high-fat diet, and these liver were compared with nonfatty livers during 48 h of normothermic ESLiP. Fatty livers demonstrated higher perfusate triglyceride, glucose, and urea production during ESLiP compared with normal livers, which reflects a higher metabolic state. In addition, a significant reduction in lipid deposits was observed on histology.86 The addition of defatting cocktail during normothermic ESLiP was also investigated in a rodent model, by Nagrath et al.88 Livers treated with a combination of defatting agents demonstrated significantly decreased intracellular lipid content and increased lipid oxidation and export, after only 3 h of ESLiP. In addition, an increase in gene expression related to lipid mobilization was observed in the treated livers as well.88
Injury to the microcirculation is a significant contributor to IRI and posttransplant dysfunction during liver transplantation.101 Liver sinusoidal endothelial cells, compared with hepatocytes, are known to be more prone to IRI during cold storage.101 In turn, the usage of prostaglandin E1 (PGE1) to improve the microcirculation has been examined by several studies. PGE1 is a potent vasodilator that also has antiplatelet and fibrinolytic effects. Hara et al90 first used PGE1 as an MPTA in an ex situ rodent model under normothermic conditions. Treated rodent livers had significantly improved bile production, in addition to decreased levels of liver injury markers: aspartate aminotransferase and alanine aminotransferase. Maida et al,91 from the same group, then corroborated these results using a rodent liver transplantation study using a similar setup. Treated rats had significantly higher survival rates, in addition to higher bile production and improved energy storage.
Prostacyclin, another naturally occurring vasodilating and antiplatelet agent, has also been examined as a potential MPTA in a porcine ESLiP model. Similar to PGE1, prostacyclin significantly improved bile production and lowered liver injury markers compared with ESLiP alone.92 Finally, 2 additional vasodilators, BQ123 (an endothelin receptor agonist) and verapamil (a calcium channel blocker), were studied in a porcine transplantation model by Echeverri et al.89 Treated livers demonstrated improved hepatic arterial flow and lower hepatocyte injury markers during ESLiP—however, no significant differences were observed 3 d after transplantation.89 This lack of significance may be due to the usage of relatively healthy donor livers, which did not undergo any warm-ischemic damage. A more significant outcome may be demonstrated in more marginal grafts.
Gene Modulation Agents
The usage of gene modulation agents, such as antisense oligonucleotides (ASOs) and siRNA in MP, is especially promising because it is much more targeted than systemic gene modulation, requiring less dosage and causing fewer side effects. Furthermore, it does not require viral transfection, which may adversely stimulate the immune system.95 Goldaracena et al93 first demonstrated the potential of using ASO to silence the virulence of HCV in a porcine ESLiP model. The ASO targeted miRNA-122, the most abundant miRNA in hepatocytes, which is a necessary factor for HCV replication. The silencing of miRNA has been shown to significantly decrease HCV activity in an in vitro model.93 Therefore, the ESLiP treatment of pretransplanted livers may have the potential to prevent HCV reinfections in patients who are HCV positive. Although this approach to prevent HCV reinfection is interesting, the high efficiency of current direct antiviral agents against HCV makes it less practical in the clinical setting.
Gillooly et al94 first demonstrated the successful siRNA uptake during ESLiP, under both normothermic and hypothermic conditions using a rodent model. The siRNA targeted the Fas receptor, whose activation contributes to a proapoptotic pathway that significantly contributes to IRI. Although the Fas pathway is only 1 potential target of gene modulation using siRNA, numerous other potential proinflammatory targets may be silenced to protect the liver.95
The same group, for example, also used siRNA targeting p53 to modulate apoptosis in an ESLiP rat model.95 Other potential targets include RelB, TNF-α, and proapoptotic caspases, which have already been demonstrated to have protective effects in murine models when delivered intravenously before the induction of ischemia.70
Other Therapeutic Agents
Two additional agents have been tested under normothermic ESLiP using rodent models. Rigo et al97 successfully demonstrated the uptake of human liver stem cell–derived extracellular vesicles during ex situ perfusion. In turn, treated livers had less histological damage and liver injury markers after 4 h of perfusion. In another study, enkephalin, a delta opioid agonist, was used during ESLiP to reduce injury caused by oxidative stress. Treated livers demonstrated significantly superior energy storage and less tissue injury markers.98
Therapeutic Agents Under Subnormothermic and Hypothermic Settings
Liu et al87 investigated using defatting cocktail in a subnormothermic ESLiP rodent model. Unlike normothermic ESLiP, subnormothermic (room temperature) ESLiP does not require temperature control and the addition of oxygen carriers, making it more practical in the clinical setting. However, the subnormothermic ESLiP time required to significantly increase perfusate lipid content was twice as long as that of normothermic ESLiP. In addition, no significant intracellular lipid content changes were observed.87 In comparison, normothermic ESLiP seems to be a more effective method of reducing steatosis.
Goldaracena et al96 used antiinflammatory agents during subnormothermic MP to minimize inflammatory damage during ESLiP in a porcine transplantation model. The authors chose a subnormothermic setting because of its inhibitory effects on Kupffer cells and inflammation compared with normothermic temperature. Although significantly lower aspartate aminotransferase and inflammatory cytokine levels were observed during ESLiP, no significant differences were observed during 3 d of reperfusion after transplantation. However, a significantly lower bilirubin level was observed in the treated groups after transplantation.
Finally, in a hypothermic ESLiP and transplantation model, Yu et al99 used mcc950, an nucleotide-binding domain leucine-rich repeat containing family pyrin domain containing 3 inflammasome inhibitor, as an antiinflammatory MPTA. Significantly lower inflammatory and injury markers were observed in treated livers after transplantation.
Kidney Machine Perfusion Therapeutics
Kidney transplant is the most effective treatment for end-stage renal failure, which affects nearly 100 000 new American patients per year.102 Deceased donor kidneys, especially DCD kidneys, are more susceptible to primary nonfunction and delayed graft function—thus the usage of hypothermic103 and normothermic104,105 ex situ kidney machine perfusion (ESKP) has been explored to protect these vulnerable kidney grafts during transplantation. Numerous functional parameters and biomarkers have been shown in NMP to be excellent predictors of kidney function during perfusion, including renal blood flow and urine output.106-108 Several biomarkers, such as glutathione S-transferase, have been examined in HMP as well.109 In addition, MPTAs have been studied in both temperatures of ESKP models; these agents have been summarized in Table 5.
Therapeutic Agents in Hypothermic Ex Situ Kidney Perfusion
MSC and its extracellular vesicles, known for their broad range of protective abilities in ESLuP (Table 1), have also been tested in a rodent ESKP model.116 After 40 min of warm ischemia, kidneys treated with MSC or its extracellular vesicles secreted lower markers of ischemic damage and glucose were in the efferent fluid, along with higher levels of pyruvate.116 This indicates an increase in energy substrate usage compared with kidneys preserved by ESKP alone. In addition, an upregulation of enzymes related to cell energy metabolism and ion membrane transport was demonstrated in the treated kidneys as well, which potentially explains the protective mechanism of MSC during ESKP.
Inhalation of carbon monoxide, a vasodilating and antiinflammatory agent, has been shown to have protective effects during kidney transplantation in animal models. Bhattacharjee et al117 created manganese-containing carbon monoxide-releasing molecules 401 to deliver carbon monoxide in a highly controlled manner. Using an ESKP porcine model, the group showed that carbon monoxide-releasing molecules 401 significantly improved renal function (increased renal blood flow and urine secretion) and lowered kidney damage markers on histology.
Microcirculatory failure due to thrombosis is a hallmark of IRI; in turn, 2 anticoagulative agents have been studied in ESKP porcine models. Sedigh et al118 treated explanted kidneys with a heparin conjugate during 20 h of ESKP. Superior kidney functions and lower kidney injury markers on histology were observed in treated kidneys. Hamaoui et al115 studied the effects of thrombalexin, a thrombin inhibitor, in both porcine and human kidney models. Treated porcine kidneys demonstrated significantly superior renal flow parameters and microvascular capillary perfusion. The treatment of 2 human kidneys demonstrated similar protective effects, in addition to lowering D-dimer and fibrinogen levels.115
Therapeutic Agents in Normothermic Ex Situ Kidney Perfusion
NMP, unlike HMP, occurs in higher metabolic state, which may allow kidney grafts to restore energy levels and heal damage caused by cold ischemic storage faster. Brasile et al114 showed that the addition of MSCs in normothermic ESKP can further accelerate the renal repair process. Using human kidney allografts, they showed that 24 h of perfusion with MSCs significantly reduced inflammatory cytokines and significantly increased adenosine triphosphate storage and various growth factors (endothelial growth factor, fibroblast growth factor 2 and transforming growth factor α).114 Sierra Parraga et al119 investigated the effect of perfusion fluid on the survival and function of MSCs. Although the perfusion fluid significantly reduced the capacity of MSCs to adhere to endothelial cells, it also increased proliferation after the cells adhere. In addition, they showed that the secretory profile of MSCs is not affected by perfusion.119 Since a large number of MSC are needed for NMP, Parraga et al investigated the impact of the freeze-thaw process on MSCs. Specifically, Sierra Parraga et al119 showed that the free-thaw process reduces survival and metabolism, increases oxidative stress, and impairs cell adherence. Pool et al113 fluorescently labeled MSCs to assess the localization and survival of MSCs during NMP in a porcine ESKP model. After 6 h of perfusion with 107 MSCs, small clusters of glomeruli showed positive staining for MSC, and these cells reached as far as the renal cortex.113
Yang et al110 explored the potential of using erythropoietin, a hormone with protective paracrine effects in the kidney, as a therapeutic agent during normothermic ESKP. In a porcine ESKP model, the addition of erythropoietin significantly suppressed inflammatory activity (caspase-3 and IL-1β) during 2 h of perfusion. Improved urine output was also observed as a result.110 The same group also examined the renoprotective effects of cyclic helix B peptide (CHBP), an erythropoietin derivative, in a similar porcine model.111 Unlike erythropoietin, CHBP does not cause erythropoiesis and its subsequential side effects. Similar to erythropoietin, CHBP improved urine output, in addition to renal blood flow and oxygen consumption.111
Tietjen et al112 recently demonstrated the potential of using anti-CD31 antibody to enhance the delivery of nanoparticles to endothelial cells of human kidney grafts. The accumulation of nanoparticles was increased by 5- to 10-fold by the addition of anti-CD31 antibodies.112 These nanoparticles can serve as “depots for long-term drug release” and can be used to deliver therapeutic agents specifically to the kidney endothelium. Other laboratories have also examined the potential of using nanoparticles to deliver antioxidative agents, antagonists to IRI mediators, and genetic material to reduce tissue damage during transplantation.120
Several other therapeutic agents have also been investigated to target specific pathways in IRI.121 Although these agents have not been tested in MP models yet, they represent promising MPTA agents in the future. Hydrogen sulfate (H2S) has known cytoprotective properties, such as mitigating oxidative stress and decreasing inflammation.122 Recently, a synthetic slow-releasing donor molecule (AP39) has been created for the delivery of H2S in organ transplant.123124 Streptokinase, a thrombolytic agent, has been shown to improve kidney microvasculature, when it is injected intraarterially into the kidneys.125126 Diannexin (a phosphatidylserine inhibitor), recombinant molecule resulting from the fusion of P-selectin glycoprotein ligand (PSGL) and human igG1(an inhibitor polymorphonuclear leukocyte recruitment), and I5NP (inhibitor of p53 expression) have also been shown to mitigate kidney IRI.127
Cardiac Machine Perfusion Therapeutics
Cardiovascular disease is currently the leading cause of death worldwide, and there are nearly 6.5 million adults in the United States who is suffering from end-stage heart failure.128 Although cardiac transplantation remains the gold standard for treating patients with heart failure, this procedure can lead to numerous posttransplant complications, such as graft dysfunction and rejection. Cardiac gene therapy has emerged as a promising approach to ameliorate IRI during transplantation and to recondition cardiac grafts to decrease the incidences of posttransplant graft dysfunction. Several studies have demonstrated the successful uptake of adeno-associated virus vector by myocytes during ex situ intracoronary administration.129130 Bishawi et al131 took this step further by administering adenoviral luciferase vectors during normothermic ex situ cardiac perfusion in a porcine transplantation model. After 2 h of NMP with intracoronary delivery of viral vectors, the donor hearts were transplanted into recipient pigs. Robust luciferase activity and protein expression were observed in all 4 cardiac chambers after 5 d of reperfusion.131
The addition of siRNA in cardiac NMP has also been explored. In a porcine ex situ perfusion model, Wei et al132 added siRNAs targeting inflammatory and apoptotic signal proteins, such as C3, nuclear factor κB-p56, caspase-8, and caspase-3 into the perfusate. Treated hearts demonstrated significantly reduced apoptosis and less structural damage compared with MP alone, in addition to improved left ventricular cardiac function.132 These successful applications of gene modulation during cardiac NMP opens the door to numerous other targeted therapies in cardiac transplantation.
The rapid advancements in MP of lung, liver, and kidney have allowed for the utilization of marginal grafts for patients who are in dire need of a lifesaving organ.133134 In turn, these advancements have also pushed for the exploration of potential therapeutic agents that may be used during the perfusion period. New advancements in MPTAs are particularly exciting because they offers several advantages compared with systemic treatment. First, they require a lower dosage of drugs to take effect; this is especially significant for drugs that require a weight-dependent dosage, such as siRNA. Second, MPTAs allow for a much more targeted method for delivering drugs with potential systemic side effects, such as MSCs and thrombolytic agents.
Currently, no MPTAs have been tested in randomized clinical trials. However, many promising MPTAs, especially in the field of lung transplantation, have been extensively tested in animal and discarded human organ models and are very close to reaching the stage of clinical trials. Some therapeutic agents, such as thrombolytic40,67 and antibacterial42 agents, have already been used to recondition discarded human grafts and led to successful transplantations. Since the success of clinical trials in kidney and liver MPs are recent, most MPTA studies in these fields are still in the animal model stage. More human studies, especially randomized trials, are needed in the future to solidify the therapeutic potential of MPTAs in these organ transplants.
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