Mitochondrial dysfunction is known to be associated with the development of acute organ diseases accompanied with inflammation, oxidative stress, and immune response.1,2 Accordingly, mitochondria are extremely sensitive to oxygen-dependent events such as ischemia, hypoxia, and reperfusion and exhibit various functions and roles depending on cellular circumstances.3,4 They are also known to initiate program cell death after substantial cellular damage.5 Indeed, numerous studies have demonstrated that mitochondrial damage and dysfunction are associated with organ damage, including lungs.6 Consequently, mitochondrial health/function can be considered as a predictor of organ quality and thus serve as a potential therapeutic target.7,8
In the organ transplant field, the importance of mitochondrial protection during preservation has been widely recognized as they play an essential role in creating ischemia/reperfusion injury and inducing cellular death.8,9 Also, mitochondria have inherent components, such as mitochondria-bound cytochrome c and mitochondrial DNA (mtDNA), acting as damage-associated molecular patterns (DAMPs), endogenous molecules known to induce proinflammatory responses including high-mobility group box 1 and extracellular ATP.10 Recently, circulating donor mtDNA has been identified as an important factor to help predict early posttransplant outcomes based on recent clinical studies,11,12 suggesting the importance of mitochondrial quality management in grafts to prevent posttransplant adverse events such as primary graft dysfunction (PGD).
Ex vivo lung perfusion (EVLP) is an effective tool to evaluate marginal grafts in current clinical practice. The contribution of EVLP on increasing the donor pool is well documented with 15%–20% expansion,13 although the impact on the posttransplant outcomes is very limited.14 Also, the EVLP system itself creates a harmful proinflammatory environment within the grafts, directly affecting graft quality with tissue mitochondrial damage and subsequent ATP depletion.15-18 In addition, elevated perfusate levels of DAMPs are associated with reduced graft transplantability and the incidence of severe PGD after transplantation.19,20
In our previous studies,18,21,22 we observed significant mitochondrial dysfunction and hypoxic damage in rat lung grafts after EVLP, indicating the quality of lung grafts can deteriorate during EVLP. Our laboratory has focused on improving the current EVLP strategy from the standpoint of graft preservation. With our original strategy to improve graft quality during EVLP, we previously found that ionized calcium (Ca2+) concentrations in EVLP perfusate were decreased compared with Ca2+ concentrations under standard EVLP settings.23 In addition to improved graft quality, these strategies conferred improved mitochondrial quality/function and attenuated inflammatory cytokine profiles in lung grafts, resulting in improved posttransplant outcomes. Consequently, these findings led us investigate the potential relationship among these factors regarding lung preservation during EVLP.
Therefore, we hypothesized that modulation of metabolic factors during EVLP may enhance graft function and overall quality resulting in improved transplant outcomes. In this study, we administered cyclosporin A (CyA), a potent inhibitor of calcineurin that controls intracellular calcium signaling, inflammation, and mitochondrial permeability transition pore (mPTP) opening, during EVLP and examined its effects on graft quality from the standpoints of preservation and transplant.
MATERIALS AND METHODS
Inbred male Lewis (RT-1l) rats weighing 250–300 g were purchased from Harlan (Harlan Sprague-Dawley Inc., Indianapolis, IN). Animals were maintained in laminar flow cages in a specific pathogen-free animal facility at the University of Pittsburgh and fed a standard diet and water ad libitum. All procedures were performed according to the guidelines of the Institutional Animal Care and Use Committee at the University of Pittsburgh and the National Research Council’s Guide for the Humane Care and Use of Laboratory Animals.
Experimental Animal Protocol
We randomly assigned animals into 3 groups: sham, control, and CyA treatment.
The lung grafts were procured and subjected to 1-hour cold ischemia. After cannulation during cold ischemia, EVLP was performed for 4 hours with and without CyA administration in perfusate (CyA and control group, respectively). After 4 hours of EVLP, lungs were maintained at 4°C for 1 hour, then an orthotopic single left lung transplantation was performed. Sham-operated animals underwent anesthesia, tracheotomy, and mechanical ventilation with 100% O2, and then the lungs were immediately removed for analysis.
EVLP in Rats
EVLP was performed using a commercially available rodent EVLP system (IL-2 Isolated Perfused Rat or Guinea Pig Lung System; Harvard Apparatus, Holliston, MA) as described previously.24 During EVLP, the lungs were ventilated with air at 37°C and perfused with Steen solution (XVIVO Perfusion AB, Göteborg, Sweden) that was deoxygenated with 6% O2, 8% CO2, and balanced N2 and was supplemented with 50 mg of methylprednisolone (Solu-Medrol; Pfizer, Inc., New York, NY) and 50 mg of cephalosporin (Cefazolin; West-Ward Pharmaceuticals Corp., Eatontown, NJ). In the treatment group, CyA (Sigma-Aldrich, St. Louis, MO) was administered into the perfusate at a final concentration of 1 μM at the time of priming. Perfusion flow was started at 10% of target flow and gradually increased for 1 hour toward a target flow rate that was calculated as 20% of cardiac output (75 mL/min/250 g donor body weight). Pulmonary artery pressure, peak airway pressure, and airway flow were monitored continuously, and partial pressure of arterial oxygen (Pao2)/fractional inspired oxygen (Fio2)ratio (P/F ratio), dynamic lung compliance, and pulmonary vascular resistance were analyzed. Every hour, the ex vivoperfused lung was ventilated with 100% O2 for 5 minutes, and then the perfusate was sampled for pulmonary oxygenation and electrolyte analysis.
Rat Orthotopic Left Lung Transplantation Following EVLP
Orthotopic, single-lung transplantation of the left lung was performed using the cuff method as described previously.22,24 After 4 hours of EVLP, the lungs were precooled with 4°C Steen solution on the EVLP system and stored at 4°C for 1 hour before transplantation. Two hours after reperfusion, the naïve lung was clamped, 100% O2 was administered for 5 minutes through a ventilator, and the recipient’s blood was sampled from the graft pulmonary vein for blood gas analysis.
Real-time Reverse-transcription Polymerase Chain Reaction
Graft tissues were collected after 4 hours of EVLP or 2 hours after transplantation following 4 hours of EVLP and assessed for mRNA levels of interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-α, and glyceraldehyde-3-phosphate dehydrogenase by SYBR Green 2-step, real-time reverse-transcription polymerase chain reaction as previously described.24
Citrate Synthase Activity Assay
Citrate synthase activity in lung tissues after 4 hours of EVLP was determined using citrate synthase activity assay kit (Sigma-Aldrich) according to manufacturer’s instruction.
ATP Content Measurement
For quantification of ATP in lung tissues after 4 hours of EVLP, an ENLITEN ATP assay system bioluminescence kit (Promega, Madison, WI) was used as previously described.22
Mitochondrial Enzyme Activity Assay
Mitochondria were isolated from lung tissues after 4 hours of EVLP using a mitochondria isolation kit (Thermo Fisher Scientific, Rockford, IL). The lung tissue was finely minced and then homogenized with cold (4°C) mitochondrial isolation buffer. Mitochondrial proteins were extracted and used for microplate assays to determine the enzyme activities of mitochondrial complex I (Abcam, Cambridge, MA) as described previously.22
Detection of Tissue Reactive Oxygen Species
Cytosolic fractions of lung tissues obtained from mitochondrial isolation were mixed with 2′,7′-dichlorofluorescin diacetate (Millipore-Sigma, Burlington, MA) for the detection of reactive oxygen species (ROS) in lung tissue after 4 hours of EVLP. 2′,7′-dichlorofluorescin diacetate is oxidized to the highly fluorescent 2′,7′-dichlorofluorescein by a variety of ROS. After incubation at 37°C, the 2′,7′-dichlorofluorescein in the sample was detected by microplate reader with the light wavelengths at 485 nm excitation and at 528 nm emission.
Mitochondrial DNA Detection in Perfusate
Total DNA was isolated from perfusate samples using DNA isolation kit (QIAamp DNA Mini Kit; Qiagen GmbH, Hilden, Germany). Following DNA extraction, real-time reverse-transcription polymerase chain reaction was utilized to amplify the mtDNA d-loop region. The absolute quantity of d-loop mtDNA was quantified based upon a standard curve generated by amplifying serial dilutions of known amounts of cytochrome c oxidase subunit 3 mtDNA.
Formalin-fixed, paraffin-embedded lung tissues 2 hours after transplantation following 4 hours of EVLP were sectioned to 4-μm thickness and stained with hematoxylin and eosin.22
Wet-to-Dry Weight Ratio
The weight of lung tissues 2 hours after transplantation were measured immediately after collection and then placed into a 60°C oven to dry for 72 hours. Tissues were weighed to determine the wet-to-dry (W/D) lung weight.
Determination of Lactate Levels in Perfusate
Lactate levels in perfusate sampled hourly during EVLP were measured using a Lactate Assay Kit II (BioVision, Mountain View, CA).24
All data were analyzed using SPSS Version 25 statistical software package (SPSS Inc., Chicago, IL). Results are expressed as mean ± SEM. Data from multiple observations over time were analyzed using 2-way repeated measures ANOVA. The data were analyzed with 1-way ANOVA followed by post hoc analysis with the Bonferroni correction for multiple comparisons. A probability level of P < 0.05 was considered statistically significant.
CyA Treatment Improved Graft Function During EVLP
Lung grafts on EVLP maintained stable function, as indicated by P/F ratio, pulmonary vascular resistance, and dynamic lung compliance, throughout 3 hours of EVLP, although lung function gradually declined over the last hour of EVLP. When CyA was administered into the perfusate, stability of all functional parameters improved throughout the 4 hours duration of EVLP (Figure 1A–C). Although perfusate electrolytes were stable during the 4 hours of EVLP, glucose levels in the perfusate dropped significantly during this time period (Figure 1D). However, CyA-treated lung grafts exhibited significantly reduced glucose consumption throughout the 4 hours of EVLP (Figure 1D). Additionally, Ca2+ concentrations were significantly lower in perfusate samples infused with CyA during EVLP compared with control samples (Figure 1E). Furthermore, perfusate acidosis developed over time on EVLP in controls but was attenuated significantly with CyA treatment (Figure 1F). As shown in Figure 1G, perfusate lactate was continuously produced by the lungs during EVLP and increased in controls, while CyA treatment significantly decreased lactate production in the lungs.
CyA Administration During EVLP Attenuated Graft Inflammation
To measure proinflammatory changes within the lungs, we next measured the mRNA expression profiles of proinflammatory cytokines in lung tissues exposed to 4 hours of EVLP. The mRNA levels of IL-6, IL-1β, and TNF-α were significantly increased in EVLP-treated lungs as compared with sham lungs that were exposed to only a short period of ventilation in vivo (Figure 2A). CyA administration of lung grafts during EVLP, however, significantly attenuated mRNA expression of IL-6 and IL-1β but not TNF-α (Figure 2A and B). For comparison, we also assayed mRNA expression profiles of proinflammatory cytokines in lung perfusate. In contrast to lung tissue profiles, concentration of IL-6 in perfusate samples after 4 hours of EVLP was reduced, although not significantly, with CyA treatment. In addition, perfusate levels of TNF-α were unaltered with CyA administration (Figure 2B).
Addition of CyA During EVLP Improved Mitochondrial Quality and Inhibited DAMP Release From Lung Grafts
To evaluate mitochondrial quality in lung grafts on EVLP with and without CyA treatment, we then measured mitochondrial density, mitochondrial complex I enzyme activity, and ATP content in lung tissues after 4 hours of EVLP. Mitochondrial density, as evaluated by citrate synthase activity, was decreased in control-treated lungs versus sham lung tissues, although mitochondrial density increased in CyA-treated lung grafts after 4 hours of EVLP (Figure 3A). Mitochondrial complex I enzyme activity in control lung tissues was also decreased compared with sham lungs after 4 hours EVLP and was significantly increased following CyA treatment (Figure 3B). In addition, tissue ATP production was attenuated in control lung grafts 4 hours after EVLP but were significantly increased in CyA-treated lung grafts to control levels during EVLP (Figure 3C).
To further confirm mitochondrial dysfunction, lung tissue ROS levels were significantly higher in controls compared with those of sham tissues after 4 hours of EVLP. These ROS levels were significantly diminished in CyA-treated lungs after 4 hours EVLP (Figure 3D). Finally, we measured circulating mtDNA in perfusate during EVLP and found the CyA administration dramatically decreased the concentration of mtDNA during EVLP compared with controls (Figure 3E).
Preconditioning of Lung Grafts With CyA on EVLP Improved Posttransplant Graft Quality
Lungs transplanted after EVLP with CyA treatment exhibited markedly improved graft function regarding P/F ratio as compared with lungs transplanted after EVLP without CyA treatment (Figure 4A). Histologically, we noted that both cellular infiltration and edema formation were remarkable in transplanted lung grafts following EVLP without CyA but were significantly attenuated in CyA-treated lung grafts (Figure 4B). After transplantation, mRNA expression profiles for IL-6, IL-1β, and TNF-α remained significantly down-regulated in CyA-treated EVLP grafts as compared with expression profiles in non-CyA-treated EVLP grafts (Figure 4C). W/D ratio of lung grafts 2 hours after transplantation was significantly increased in controls compared with pretransplanted grafts. W/D ratio in grafts with pretreatment of CyA during EVLP was considerably less than control grafts (Figure 4D).
CyA As a Graft Protection Agent During EVLP
In organ transplantation, mitochondrial protection is considered crucial given that mitochondria, as primary O2 sensors, are extremely susceptible to ischemia-reperfusion injury. With current standard graft preservation also known as cold static preservation, the cold ischemia is considered a “priming” phase that can unnecessarily deplete cells of Ca2+ and ATP, altering mitochondrial membrane sensitivity and depolarizing their membrane potential.25,26 Upon reperfusion, mitochondrial permeability transition triggers both mitochondrial ROS generation and opening of mPTP, ultimately leading to DAMP release and tissue inflammation. Undoubtably, mPTP opening is widely known in mediating the mitochondrial response to ischemia/reperfusion injury and initiation of apoptosis.25
CyA has been well-documented to prevent ischemia-reperfusion injury in a multitude of organs including heart, liver, and kidney.27,28 However, in the setting of lung transplantation and EVLP, the potential beneficial effects of CyA are largely unknown. Indeed, PGD remains a leading cause of morbidity and mortality in posttransplant lung recipients.29 We have previously confirmed mitochondrial injury in lungs during EVLP induced by oxidative stress, inflammation, and hypoxia.18,21,22 Such damage and dysfunction are known to negatively impact posttransplant outcomes.30-32 Here, we present evidence that CyA (1 μM) treatment of lung grafts on EVLP improved functional and physiological parameters. In addition, our results strongly demonstrate that treatment of EVLP lungs with CyA favorably affects mitochondrial function/biogenesis and attenuates graft proinflammatory responses.
A remarkable finding of the present article is the demonstration that CyA treatment significantly reduced ionized Ca2+ concentrations in EVLP perfusate over time (Figure 1E). Calcium is known to play an important role in intracellular signaling and cellular homeostasis.33 Although the exact dynamics of intracellular calcium flux in lungs on EVLP are largely unknown, our data regarding increased mitochondrial dysfunction in non-CyA-treated lungs suggest elevated Ca2+ release from mitochondria. Specifically, higher concentrations of Ca2+ in perfusate were observed throughout 4 hours of EVLP suggesting mitochondrial damage may be initiated at an early time point. We previously reported that perfusate Ca2+ elevation was attenuated using our previous EVLP strategies, which consequently improved mitochondrial quality.23 More specifically, by sequestering and releasing Ca2+, mitochondria serve as important regulators of cellular Ca2+ as well as effectors of mitochondrial metabolism, ATP production, and cell death.12 Thus, we believe elevated Ca2+ concentrations in perfusate can be a contributing and reliable marker of mitochondrial damage in grafts during EVLP.
CyA May Prevent Secondary Lung Inflammation Through DAMP Suppression
Circulating mtDNA, released from apoptotic and necrotic cells, is a well-known DAMP that is recognized by pattern recognition receptors such as toll-like receptors.34 This interaction can activate proinflammatory pathways of the local immune system, spreading inflammation to surrounding and remote tissues via delivery of DAMPs through circulating blood. Following critical cell injury, release of mtDNA is facilitated via the mPTP with sustained opening resulting in mitochondrial dysfunction, organelle swelling, rupture, and ultimate cell death.
Regarding lung transplantation, a recent retrospective study by Scozzi et al11 found that circulating mtDNA is associated with PGD and that lung recipients before transplantation had slightly increased mtDNA levels compared with healthy controls. These data suggest that donor mtDNA has a critical role in determining graft tissue injury and that increased mtDNA in donor circulation could be considered as a consequence of graft quality. The circulating mtDNA in donor organs may not function as DAMP in recipients because they can be washed out from grafts during procurement procedure; however, these liver, kidney, and lung evidences commonly found higher whole intact mitochondria in plasma from deceased organ donors compared with those in healthy controls along with the finding of circulating mitochondrial DAMPs.11,35 Additionally, a recent study by Lin et al36 using a mouse heart transplant model demonstrated this circulating intact mitochondria can activate the endothelial cells of donor tissues and their production of inflammatory cytokines and chemokines, resulting in increased allograft rejection. Taken together, observations of mtDNA in circulation may indicate coexistence of whole intact mitochondria, which may increase the sensitivity of endothelial cells in grafts and may increase the incidence of posttransplant complication and rejection after transplantation. In this study, we showed an overall increase in cell-free mtDNA content in EVLP perfusate with a steady decrease over time in control animals (Figure 3E), suggesting mtDNA is released at an early time point (consistent with our findings of increased Ca2+ release) and may contribute to inflammatory responses through its binding to tissue-specific receptors or circulating inflammatory cells. In an “acellular” EVLP setting, CyA may effectively diminish mtDNA release from lung tissue via inhibiting mPTP opening, thereby protecting the lungs from proinflammatory insult and leading to both improved graft EVLP preservation and posttransplant outcomes. More specifically, our findings emphasize the importance of mitochondrial protection in an "acellular" EVLP setting, which is devoid of RBCs, thus potentially preventing additional lung injury and compromised graft quality. Although not directly relevant to our studies, it is interesting to note that Hotz et al37 recently identified an unfamiliar function for RBC as in vivo scavengers of cell-free mtDNA, perhaps providing a natural in vivo mechanism to alleviate lung injury during diseased states.
The Effect of CyA on Graft Metabolism During EVLP
Interestingly, we observed CyA administration during 4 hours EVLP inhibited glucose consumption of graft tissues in a time-dependent manner (Figure 1D). Both clinical38-41 and animal17,42-44 studies have demonstrated that while on EVLP, lung allografts consume increased amounts of glucose and produce lactate. It is known that inflammation can be a trigger of glycolysis in the lungs and the multiple types of cells including parenchymal and nonparenchymal cells are considered as glucose consumers though the major contributor(s) of glucose uptake during EVLP is unknown.45 Thus, such lung metabolic activity may serve as a potential biomarker to monitor the graft condition including edema development and inflammation during EVLP.42 Using a rat EVLP model, we have previously demonstrated that this hyper-glycolysis can trigger a hypoxic response based on findings with “dual EVLP” and optimal perfusate oxygenation.21,22 More importantly, we found that mitochondrial dysfunction was minimized in lung grafts treated via these strategies. Taken together, these data suggest that overall glucose consumption of grafts may be associated with mitochondria damage and graft oxygenation status. Similar findings have been reported previously in a hypoxia-induced pulmonary hypertension rat model,46 potentially due to direct CyA effects on modulating mitochondrial metabolism47 and/or cellular oxygen consumption in ischemic/hypoxic tissue.47,48 Despite improvement of graft metabolism following CyA treatment during EVLP, further investigations are required to ascertain if CyA alters cellular metabolism directly or indirectly via mitochondrial protection.
Our studies using a rat lung transplant model have several potential limitations as described previously. Regarding this study, CyA is well known as an immunosuppressant and its effects are based on inhibition of calcineurin signaling in T cells.49 Based upon this current study, we acknowledge that we do have limited data about the effects of CyA on immune suppression in the lungs during EVLP. It is widely recognized that the standard procurement procedure using Perfadex is unable to extract entire nonparenchymal cells and blood from lung tissue, which creates microchimerism in the recipient after transplantation,50 and the circulating cells can be detected in perfusate during EVLP.16,51-53 Consequently, partial effects of CyA demonstrated in this study may result from its tissue residual T-cell suppression rather than its apparent mitochondrial protection. Additionally, the dose of CyA (final perfusate concentration of 1 μM) utilized in this study was based upon previous literature which suggested that higher doses (>1 μM) of CyA can be harmful to lungs on EVLP.54 Future dose-response studies are planned that will help elucidate the potential beneficial effects of CyA regarding EVLP graft quality and preservation.
During EVLP, CyA administration can have a preconditioning effect through both its anti-inflammatory and mitochondrial protective properties, leading to improved lung graft preservation, which may result in enhanced graft quality after transplantation. Further studies are necessary to ascertain the therapeutic potential of CyA in limiting metabolic insult to lungs during EVLP for improved clinical outcomes.
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