Lung transplantation (LTx) is a life-saving treatment for patients with end-stage pulmonary disease. A major threat to early graft function and recipient survival after LTx is ischemia–reperfusion injury (IRI), which is a major cause of primary graft dysfunction (PGD),1-3 and significantly increases the risk of acute rejection and long-term graft dysfunction.4 Hypothermic organ storage is associated with a cascade of cellular events such as oxidative stress that result in release of proinflammatory mediators for leukocyte activation during reperfusion.2,5 Restoration of the blood flow to the ischemic lung after implantation initiates a series of cellular and molecular alterations, known as IRI that eventually leads to pulmonary dysfunction.6,7 To date, no effective therapy is available to prevent IRI after LTx. One promising modality is ex vivo lung perfusion (EVLP), which not only has emerged as a valuable tool to evaluate graft function before LTx, but also it provides a platform to treat the graft with pharmacological agents or molecular interventions such as gene therapy.8-10
Recently, short noncoding RNA (microRNA) was found to provide new targets for novel gene therapy.11-14 Interestingly, 1 microRNA (miR) can regulate several different mRNA, hence modulating miR expression can potentially modify entire gene networks.14,15 Key miR has been discovered to regulate numerous target mRNA during IRI.14 Even though it is still experimental, modulating the miR expression has been shown to ameliorate IRI in cardiac, renal, hepatic, and neuronal tissues.16-19 Additionally, miR was found to regulate the signaling pathways of inflammatory and immunological responses of pulmonary diseases.13,20-22 There has been growing interest in studying the role of miR in acute lung injury, PGD, and chronic rejection.23-26 In this pilot study, we aim to examine the feasibility of using the EVLP platform to study miR expression in human lungs and test the hypothesis that miR signature changes in response to cold ischemia and ex vivo reperfusion (CI/EVR).
MATERIALS AND METHODS
Ex Vivo Perfusion of Human Lungs
Before clinical application, our group used the EVLP platform to establish a biorepository of human lung samples for translational research. The work in the current study was conducted retrospectively on the biobank stored samples from 24 deceased donors that did not meet the ideal criteria for LTx, whereas clinical EVLP was not available at the time. Marginal criteria included suboptimal PaO2/fraction of inspired oxygen (PaO2/FiO2) ratio or abnormal chest X-ray in addition to unavailability of suitable recipient. Lungs with severe pulmonary infection, severe lung contusion, and severe emphysema were excluded (EVLP donor characteristics listed in table S1, SDC, http://links.lww.com/TP/B965).
Procurement of the lung grafts was arranged through our local Organ Procurement Organization (Lifebanc) according to an exempted research protocol using the standard surgical technique for clinical LTx (IRB#11-737). Directly after procurement, the lungs were preserved in a semi-inflated state with cold pulmonary artery perfusion using Perfadex solution (XVIVO Perfusion Inc., Denver, CO). Cold ischemia time was defined as the time between crossclamping and the start of EVLP (median 6, IQR 5–13 h). The donor’s lungs were perfused using cellular EVLP for 2 h with 100% of estimated cardiac output and tidal volume of 6 mL/kg with positive end-expiratory pressure of 5 cmH2O based on the donor ideal body weight, as previously described.27 The EVLP system was primed with 2.0 L of STEEN solution, heparin 10 000 IU, and packed red blood cells 500–600 mL, whose hematocrit level was 10%–15% as previously reported.28 The pH in the solution was adjusted with isotonic trometamol. Imipenem (100 mg) was added to the perfusate. The cellular EVLP protocol utilized in this study is designed to simulate the physiologic changes associated with reperfusion of the lung graft after clinical transplantation.
Samples of the perfusate solution were collected at 3 time points: at the initiation of EVLP (0 h), after 1 h, and 2 h of EVLP. Lung tissue samples were obtained at 2 time points: (1) at the end of the CI time and before initiating EVLP (ischemia group) and (2) at the end of 2-h EVLP (reperfusion group). As the cold ischemic effect is expected to be uniform in all areas of the lungs, although reperfusion response is expected to be more significant in the lower lobes, all the reperfusion group samples were collected from the anterior lower edge of either lower lobe. Additionally, to avoid manipulation of the lower lobes microenvironment by biopsies before initiating the EVLP, all the ischemia group samples were collected from the anterior lower edge of the right middle lobe. Each sample was removed as 2–3 cm peripheral wedge by a stapler. Samples were either frozen immediately after retrieval in liquid nitrogen and stored in –80°C freezer or fixed in formalin.
Characterization of EVLP-associated Inflammatory Response
Samples of the EVLP perfusate solution were evaluated in duplicate for 5 cytokines (IL-1β, IL-6, IL-8, IL-10, and TNF-α) at 0, 1, and 2 h of EVLP. The cytokines were run in multiplex using the Luminex Platform (R&D Systems Inc., Minneapolis, MN). Samples were compared to their curves based upon the linear range defined by the standards and the low and high-quality controls. Formalin-fixed, paraffin-embedded lung tissue was sectioned at 5-μm, stained with hematoxylin and eosin, and examined for pathological changes under light microscopy. Quantification of inflammation was assessed through area fraction (percentage of pixels in the selected area) of inflammatory cells infiltration using ImageJ software.29
As our EVLP research protocol did not include sampling before CI, there were no available control lung samples (without exposure to CI or EVR) in our biobank. We obtained control of lung samples (n = 6) from the Pulmonary Hypertension Breakthrough Initiative Research Network, as previously described.30 Briefly, donor lungs that were not utilized for clinical transplantation were inflated with normal saline via the bronchial tree, cut sagittally into slices, and then immersed in RNAlater before freezing at –80°C (control donor characteristics listed in Table S2, SDC, http://links.lww.com/TP/B965).
MicroRNA Expression Profiling
Tissue samples from the 3 groups (control, ischemia, and reperfusion) were used for miR expression profiling by next-generation sequencing technology, which is a powerful approach for both profiling and discovering novel miRs.31 Total RNA was extracted using the miRNeasy RNA isolation kit (Qiagen Inc., MD). Bioanalyzer was used for RNA quality control. Small RNA libraries were prepared using the Illumina Small RNA library preparation kit (Illumina, Inc. CA). Samples were barcoded to allow multiplexing, then pooled, and run on the MiSeq next-generation sequencer (Illumina, Inc. CA). Sequences were quality trimmed and then aligned to human miRbase version 21 to identify the miR species. The microarray was performed on available tissue samples from 8 EVLP cases after CI (n = 8) and all 24 EVLP cases after EVR (n = 24) in addition to control samples from 6 donors (n = 6).
Differential expression analysis of the miR sequencing data was through HTSeq and DESeq2 software package. Read counts for each miR were compared between the 3 groups (ischemia versus control and reperfusion versus control) to identify those that show significant change during ischemia and after reperfusion. Downstream analysis was performed using available online human genome database. “TargetScanHuman, Release 7.2”32 was used to identify the expected target genes regulated by the key miRs with validated significant expression change, as well as predicted interactions. For predicted targets network analysis, STRING 11.0 webserver was utilized.33
Validation of miR Expression Using Quantitative Polymerase Chain Reaction
The extracted RNA from the samples using Taqman microRNA assays (Invitrogen Inc., CA) was normalized to RNU48, according to the manufacturer’s instructions. The miR expression was compared between the 3 groups (ischemia versus control and reperfusion versus control).
Expression of the microRNAs with the most significant fold change in the microarray was examined in available lung tissue samples from 8 EVLP cases after ischemia (n = 8) and 17 EVLP cases after reperfusion (n = 17) in addition to control samples from 6 donors (n = 6).
In Situ Hybridization Detection of miR-17 and miR-548b
Lung tissue samples from the 3 groups were fixed in formalin and embedded in paraffin. Locked nucleic acid probes hsa-miR-17, hsa-miR-548, and Scramble-miR, were prepared according to the manufacturer’s recommendations (Qiagen Sciences Inc., Germantown, MD). The in situ hybridization (ISH) optimization for miR-17 and miR-548 detection was done according to a previously published protocol.34 Optimal conditions were: pretreatment with proteinase K for 4 min and probe concentration of 25 pmoles per 100 microliters of probe cocktail. Slides were tested blinded to experimental conditions. Negative controls included scrambled probe and omission of probe. Colorectal grade II adenocarcinoma cells were tested as positive control (Discovery Life Sciences, Los Osos, CA). Quantification of miR-17 and miR-548b positivity (number of positive pixels/total number of pixels) in the images were analyzed using ImagScope software (Aperio Technologies, Inc., Vista, CA).
The sample size within each group was based on sample availability and statistical test power. For the microarray analysis, significant miR expression changes were defined by an adjusted probability cutoff of q ≤ 0.05 (false-discovery rate) and if the fold change was >2. For cytokines array and quantitative polymerase chain reaction (qPCR), a Student’s t-test was used to analyze the difference between groups. All data were expressed as mean ± SE unless otherwise specified, P < 0.05 were considered significant.
Cellular EVLP-associated Inflammatory Response
In this study, we used our cellular EVLP system as a model to study human lung response to CI and ex vivo reperfusion. Examination of the change of cytokines levels in the EVLP perfusate (n = 13) showed significant increase of inflammatory cytokines production (IL-6, IL-8, IL-10, and TNF-α) in a time-dependent manner compared to baseline (at 1 h and 2 h compared to 0-h EVLP, P < 0.001) (Figure 1). The levels of IL-1β were significantly higher only at 2 h but not at 1-h EVLP (P = 0.09 and 0.01, respectively). Histological analysis has shown patchy infiltration of inflammatory cells in the lung tissues of the ischemia and reperfusion groups compared to the control group (Figure S1, A, SDC, http://links.lww.com/TP/B965). Quantification of inflammation showed increased inflammatory cells infiltration after exposure to CI (P = 0.08) and EVR (P = 0.01), Figure S1, B, SDC, http://links.lww.com/TP/B965.
Small RNA Sequencing Analysis
In order to examine the change in the human lung miR expression profiling in response to CI/EVR, comparative analysis of the next-generation sequencing data was performed which showed a total of 21 miR with significant expression change (adjusted P < 0.05) was detected in the ischemia group when compared to the control group, of which 7 miR were upregulated and 14 miR were downregulated (Figure 2). A total of 47 miR with significant expression change were detected in the reperfusion group when compared to the control group, of which 25 miR were upregulated and 22 miR were downregulated. There were 15 miR with the same pattern of expression in both ischemia and reperfusion groups (Figure 2). Sequencing data analysis did not detect miR with significant expression change between ischemia versus reperfusion group.
The top 4 miR with the most significant fold change after CI and EVR (miR 29a, miR-17, miR548b, and miR-628) were validated using qPCR. We found significant higher expression of miR-17 in ischemia versus control groups (P = 0.04), and reperfusion versus control group (P = 0.03). There was a significant higher expression of miR-548b in reperfusion group versus control group (P = 0.04), but not in ischemia group versus control group (P = 0.12), Figure 3A and B. There was no significant statistical difference in miR-29a expression between ischemia group versus control group (P = 0.08) or reperfusion group versus control group (P = 0.24). Similarly, there was no significant statistical difference in miR-628 expression between ischemia group versus control group (P = 0.34) or reperfusion group versus control group (P = 0.07), Figure 3C and D.
Detection of miR-17 and miR-548b in Lung Tissues
ISH studies showed increase expression of both miR-17 and miR-548b in the airway alveolar epithelial cells in the ischemia and reperfusion groups, compared to the control group (Figure 4A and B). Quantification of positivity in the images showed significant increase of miR-17 expression in the ischemia and reperfusion groups, compared to the control group (P < 0.0001 and <0.001, respectively), Figure 4C. Quantification of positivity in the images showed significant increase of miR-548b expression in the ischemia and reperfusion groups, compared to the control group (P < 0.001 and <0.01, respectively), Figure 4D. Results of positive and negative controls are showing in Figure S2A and B, SDC, http://links.lww.com/TP/B965. Figure S2C, SDC, http://links.lww.com/TP/B965 illustrating the quantification of miR-17 and miR-548 positivity in the tissues.
Downstream Pathway Analysis
We examined the predicted target genes for miR-17 and miR-548b using online human genome database “TargetScanHuman, Release 7.2.”32 For miR-17, 4731 target genes were found, of which 22 are interleukin-, 8 cytokine-, 22 TNF-, 22 immune-, 18 apoptosis-, 12 chemokines-, 5 Toll-like receptor (TOLL)-, and 7 hypoxia-inducible factor (HIF)-related genes. For miR-548b, 3205 target genes were found, of which 7 are interleukin-, 5 cytokine-, 11 TNF-, 12 immune-, 6 apoptosis-, 10 chemokines-, 3 TOLL-, and 1 HIF-related genes. Both miR-17 and miR-548b share 5 interleukin-, 3 cytokine-, 7 TNF-, 2 immune-, 5 apoptosis-, 1 chemokines-, 3 TOLL-, and 1 HIF-related mutual target genes (Table S3, SDC, http://links.lww.com/TP/B965). Predicted interactions between miR-548b and target genes related to lung injury are listed in Table S4, SDC, http://links.lww.com/TP/B965. Predicted interactions between miR-548b and target genes related to lung injury are listed in Table S5, SDC, http://links.lww.com/TP/B965. Network analysis of miR-17 and miR548b predicted targets as well as regulatory proteins of miR-mediated functions is shown in Figure 5, which showed high degree of connectivity among these proteins.
Response to Ischemia and Reperfusion Using EVLP Platform
EVLP is a recent modality developed to assess allograft function before LTx, which provides an excellent platform not only to study human lung ischemia–reperfusion response but also to test novel therapeutics.35-38 Yeung et al have successfully delivered IL-10 gene therapy in porcine LTx model with improvement of graft function.10 The EVLP technology includes ventilation and perfusion of the allograft in a closed ex vivo circuit under normothermic conditions using cellular preservative solution and 100% cardiac output of the donor ideal body weight “Lund protocol.”36,37 Nilsson et al have shown increase in lung weight/edema and decrease compliance after 1 and 4 h of cellular EVLP.39 In the same porcine model, expression of inflammatory cytokines IL-6, IL-8, IL-10, and TNF-α was upregulated in the lung tissue after 1 h of cellular EVLP.
Similarly, in our human EVLP model, production of inflammatory cytokines IL-1β, IL-6, IL-8, IL-10, and TNF-α was significantly increased in the perfusate solution after 1 and 2 h of reperfusion.40 Histology of lung tissue samples showed patchy infiltration of inflammatory cells after ischemia and reperfusion, which simulate radiologic findings of PGD in the clinical settings with nonspecific reticular opacities pattern in chest X-ray.41 These results demonstrate the development of reperfusion response of human lungs with initiation of cellular EVLP. However, the EVLP platform is a closed circuit with no exposure to continuous recruitment of inflammatory/immune cells or cytokines from the bloodstream as in a recipient LTx patient. In a porcine lung EVLP model, Nilsson et al have shown that the surge of inflammatory cytokines after 1 h will decrease if reperfusion continues for 4 h.39 Additionally, the blood used in perfusion passes through leukocyte filter within the EVLP circuit. Subsequently, the ischemia-reperfusion state in the lung tissues after 2 h of EVLP is expected to be secondary only to the resident cell activation after reperfusion, such as alveolar epithelial cells, endothelial cells, and alveolar macrophages.
MicroRNA Signature of Human Lung Ischemia-reperfusion
MicroRNA is a short noncoding RNA that was found to function as a critical regulator of gene expression in different physiological and pathological processes, including inflammatory and immune responses.13,14 It is largely acknowledged the miRs function as negative gene expression regulators in the cytoplasm.42-44 However, recent evidence has shown that miRs can, directly or indirectly, upregulate gene expression through nuclear function.42-44 The interaction of miR with their target genes is dynamic and reliant on several factors, such as subcellular location of miR, the abundancy of miR, and target mRNA.44 The role of miR has been investigated in acute lung injury, PGD, and chronic allograft rejection.23-26 Nevertheless, the data of the former studies were generated either from animal models or nonexperimentally controlled clinical samples. In our study, using an ex vivo lung model of human lung ischemia-reperfusion gives the advantage of controlled experimental settings along with generating direct clinically relevant data. Similar approach was followed by Crawford et al to study the miR signature of human lung IRI using EVLP platform.45 Their approach included 4 h of EVLP and sample collection every hour. In their microarray data analysis, 7 miR had log2 fold change >1.0 or <−1.0; 4 were downregulated (miR-135A, miR-143, miR-145, and miR-1284), whereas 3 miR were upregulated (miR-17, miR-146a, and miR-155) during reperfusion compared to baseline 0 h (after cold ischemia).45 Similarly, in our microarray data analysis, miR-135A, miR-145, and miR-1284 were downregulated, and miR17, miR146a, and miR-155 were upregulated after 2 h of EVLP. However, miR-143 was upregulated in our microarray data. Additionally, only miR-17 in our microarray data has significant expression change (adjusted P < 0.05) after reperfusion compared to control lungs without CI or EVR, which was validated by qPCR. These differences in findings could be related to the heterogeneity between donor lung conditions, duration of cold ischemia, biopsy location, EVLP protocol, and study control. We also found that miR-548b was significantly upregulated after reperfusion compared to control. Xu et al studied miR expression profiling in patients with chronic lung rejection and they found that miR-548d plays an important role in development of bronchiolitis obliterans syndrome after LTx through affecting TGF-b and B cell receptor signal pathways.25 Interestingly, Ladak et al were able to modulate TGF-β1– induced injury in human bronchial epithelial cells through manipulating miR-200b-3p expression, which could provide a potential therapy against airway injury.46
Role of miR-17 and miR-548b in Human Lung IRI
In response to CI/EVR, human lung alveolar epithelial cells expressed miR-17 and miR-548b, as shown in the ISH assays in our study, which is a novel finding. The alveolar epithelial cells are recognized as important mediators of lung IRI via the release of proinflammatory cytokines and chemokines.5,6 They are one of the reisdent cells site for generation of reactive oxygen species in response to ischemia-reperfusion cascade.5,47 More importantly, IRI induces apoptosis of the alveolar epithelial cells causing disruption of the alveolar-capillary barrier with subsequent increased microvascular permeability and pulmonary edema.48,49 The percentage of apoptotic cells after reperfusion in clinical LTx was found not to be associated with magnitude of the postoperative graft dysfunction and worse outcomes.50 In a murine model of acute lung injury, Zhaojun et al showed that downregulation of miR-17 may promote apoptosis of alveolar epithelial cells through overexpression of FoxA1.23 Additionally, miR-17 and miR-548 clusters have been shown to regulate cellular proliferation in lung cancer.51-53 This could explain the increased expression of both miRs in the alveolar epithelial cells after CI/EVR in our model.
On the other hand, miR-17 has been shown to regulate alveolar macrophage inflammatory response and lung epithelial cell-derived macrophage migration.54,55 Oglesby et al showed that overexpression of miR-17 in cystic fibrosis airway epithelial cells decreases proinflammatory interleukin-8 production.56 Liang et al have performed a wide-genome analysis of miR-548 family that identified target genes for Leukocyte transendothelial migration, Natural killer cell-mediated cytotoxicity, Cytokine–cytokine receptor interaction, B cell receptor signaling pathway, and TGF-beta signaling pathway.57 Fesen et al have shown that miR-548 was overexpressed in patients with chronic cystic fibrosis infection.58 Downstream analysis for miR-17 and miR-548b has identified an abundant of target genes related to lung injury. Interestingly, the 2 miRs share a number of mutual targets, which could suggest that miR-17 and miR-548b may interact at some level in the signaling pathway and potentially provide novel therapeutic targets. A number of inflammatory mediators governed by these target genes have been shown in prior studies to be associated with lung IRI after transplantation (Table 1).
This is a pilot study to test the feasibility of using EVLP to examine miR expression change in human lungs. The study was conducted in a retrospective manner utilizing an established biobank of human lung tissues and based on the availability of the sample in each study group. Following a certain approved protocol for human lung EVLP at our institution, there was no tissue sampling available before cold ischemia or reperfusion. The control group was used as reference baseline for human lung miR profiling without exposure to cold ischemia or reperfusion injury. Also, there was no direct reperfusion group without prior exposure to cold ischemia to include in the analysis. Despite these limitations, the preliminary data presented provide valuable information for future research.
In summary, in this pilot study, the EVLP platform was proven to be a feasible model to study miR expression change in human lungs after exposure to CI/EVR. Our data have shown that human lung alveolar epithelial cells express miR-17 and miR-548b in response to CI/EVR. Both miR have abundant expected target genes related to IRI cascade. These preliminary findings are novel and warrant further investigation to explore the downstream pathway for miR-17 and miR-548b in alveolar epithelial cells. Ultimately, developing a molecular therapy that targets a critical regulatory step of lung IRI cascade during EVLP, may promise an effective treatment to reduce or prevent PGD after LTx.
We thank Tracey Bonfield, PhD from CWRU for performing the cytokines analysis. We thank Gerard J. Nuovo, MD, and Adel Mikhail, PhD from Phylogeny Inc. for performing the in situ hybridization assays of the microRNA. This work is supported by RPC#175 funding award from Lerner Research Institute at Cleveland Clinic to H.E. Control group tissue samples were provided by the Pulmonary Hypertension Breakthrough Initiative (PHBI) Research Network. Funding for the PHBI is provided under an NHLBI R24 grant, #R24HL123767, and by the Cardiovascular Medical Research and Education Fund (CMREF).
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