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The Journal of Heart and Lung Transplantation
International Society for Heart and Lung Transplantation.

Mitochondrial transplant after ischemia reperfusion promotes cellular salvage and improves lung function during ex-vivo lung perfusion

Open AccessPublished:January 11, 2023DOI:https://doi.org/10.1016/j.healun.2023.01.002

      Background

      In lung transplantation, ischemia-reperfusion injury associated with mitochondrial damage can lead to graft rejection. Intact, exogenous mitochondria provide a unique treatment option to salvage damaged cells within lung tissue.

      Methods

      We developed a novel method to freeze and store allogeneic mitochondria isolated from porcine heart tissue. Stored mitochondria were injected into a model of induced ischemia-reperfusion injury using porcine ex-vivo lung perfusion. Treatment benefits to immune modulation, antioxidant defense, and cellular salvage were evaluated. These findings were corroborated in human lungs undergoing ex-vivo lung perfusion. Lung tissue homogenate and primary lung endothelial cells were then used to address underlying mechanisms.

      Results

      Following cold ischemia, mitochondrial transplant reduced lung pulmonary vascular resistance and tissue pro-inflammatory signaling and cytokine secretion. Further, exogenous mitochondria reduced reactive oxygen species by-products and promoted glutathione synthesis, thereby salvaging cell viability. These results were confirmed in a human model of ex-vivo lung perfusion wherein transplanted mitochondria decreased tissue oxidative and inflammatory signaling, improving lung function. We demonstrate that transplanted mitochondria induce autophagy and suggest that bolstered autophagy may act upstream of the anti-inflammatory and antioxidant benefits. Importantly, chemical inhibitors of the MEK autophagy pathway blunted the favorable effects of mitochondrial transplant.

      Conclusions

      These data provide direct evidence that mitochondrial transplant improves cellular health and lung function when administered during ex-vivo lung perfusion and suggest the mechanism of action may be through promotion of cellular autophagy. Data herein contribute new insights into the therapeutic potential of mitochondrial transplant to abate ischemia-reperfusion injury during lung transplant, and thus reduce graft rejection.

      Abbreviations:

      PGD (primary graft dysfunction), ROS (reactive oxygen species), IRI (ischemia-reperfusion injury), EVLP (ex-vivo lung perfusion)
      Lung transplantation remains the sole curative treatment in numerous end-stage lung diseases. However, because of the limited supply of transplantable lungs, a quarter of patients succumb to disease while awaiting an organ.
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      • Smits JM
      • Persijn GG
      • Haverich A
      Listing for lung transplantation: life expectancy and transplant effect, stratified by type of end-stage lung disease, the Eurotransplant experience.
      Further, clinical survival following transplant is reduced by the incidence of primary graft dysfunction (PGD). Impacting 30% of all lung transplant recipients,
      • Diamond JM
      • Arcasoy S
      • Kennedy CC
      • et al.
      Report of the International Society for Heart and Lung Transplantation working group on primary lung graft dysfunction, part II: epidemiology, risk factors, and outcomes-A 2016 consensus group statement of the International Society for Heart and Lung Transplantation.
      PGD causes 64.9% mortality within the first year following a transplant, compared to 20.4% without PGD.
      • Christie JD
      • Kotloff RM
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      • et al.
      The effect of primary graft dysfunction on survival after lung transplantation.
      Thus, therapeutics designed to curb the development of PGD are in high demand.
      Several risk factors can contribute to PGD, including donor health, procurement technique, tissue ischemia-reperfusion injury (IRI), and recipient health.
      • Porteous MK
      • Lee JC
      Primary graft dysfunction after lung transplantation.
      Of these, IRI represents the most predictable factor due to standardized cold ischemic preservation protocols. Therefore, targeting mechanisms of lung injury induced by IRI may be a choice route toward combatting PGD. In multiple organ systems, mitochondrial health is a critical factor in tissue recovery following IRI damage.
      • Saikumar P
      • Dong Z
      • Weinberg JM
      • Venkatachalam MA
      Mechanisms of cell death in hypoxia/reoxygenation injury.
      Mitochondrial dysfunction during ischemia impairs ATP generation and ion-channel function, leading to mitochondrial swelling and cellular edema.
      • Ferrari RS
      • Andrade CF
      Oxidative stress and lung ischemia-reperfusion injury.
      Reperfusion then results in the formation of mitochondrial-derived reactive oxygen species (ROS), notably in the pulmonary endothelium and tissue-resident immune cells.
      • Ferrari RS
      • Andrade CF
      Oxidative stress and lung ischemia-reperfusion injury.
      Excessive ROS can cause pro-inflammatory signaling and cell death, culminating in activation of antigen-presenting cells in donor tissue and acute rejection in the recipient.
      • Land WG
      The role of postischemic reperfusion injury and other nonantigen-dependent inflammatory pathways in transplantation.
      Indeed, mitochondrial impairment, upstream of ROS formation, is suggested to be a causative event in the resulting IRI-mediated cellular damage.
      • den Hengst WA
      • Gielis JF
      • Lin JY
      • Van Schil PE
      • De Windt LJ
      • Moens AL
      Lung ischemia-reperfusion injury: a molecular and clinical view on a complex pathophysiological process.
      Supporting data demonstrate that disruption of mitochondrial membrane potential following IRI preceded apoptotic signaling and cellular damage in rat lungs.
      • Sommer SP
      • Sommer S
      • Sinha B
      • et al.
      Ischemia-reperfusion injury-induced pulmonary mitochondrial damage.
      Further, preconditioning treatments that reduced IRI-associated mitochondrial damage prevented the ensuing endothelial cell impairment and partially reversed immune cell infiltrates in rat lung.
      • Sommer SP
      • Sommer S
      • Sinha B
      • et al.
      Glutathione preconditioning ameliorates mitochondria dysfunction during warm pulmonary ischemia-reperfusion injury.
      Together, these data provide a case for targeting mitochondrial repair to manage injurious lung IRI.
      Intercellular mitochondrial transfer provides an exogenous mitochondrial source, replenishing dysfunctional mitochondria in recipient cells.
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      • Vacca RA
      • Moro L
      • Atlante A
      Mitochondria can cross cell boundaries: an overview of the biological relevance, pathophysiological implications and therapeutic perspectives of intercellular mitochondrial transfer.
      The replacement of native mitochondria with freshly isolated, respiration-competent mitochondria following a 2-hour ischemic period improved murine lung function and decreased immune cell infiltration.
      • Moskowitzova K
      • Orfany A
      • Liu K
      • et al.
      Mitochondrial transplantation enhances murine lung viability and recovery after ischemia-reperfusion injury.
      These data provide a case for the use of mitochondrial transplant to combat pulmonary IRI.
      The use of ex-vivo lung perfusion (EVLP) technology affords the opportunity to treat and improve marginal lungs without direct drug administration to the transplant recipient. Herein, we sought to utilize the EVLP platform to recover IRI-induced lung injury through mitochondrial transplant. To that end, we developed a novel method to freeze and store isolated mitochondria from porcine heart tissue. These intact, functional mitochondria improved cellular health and viability through promotion of the autophagy nucleation pathway MEK, thus improving lung function. These data suggest that mitochondrial transplant serves to improve the underlying mitochondrial dysfunction associated with IRI and contribute new insights into the therapeutic potential of mitochondrial transplant in PGD.

      Methods

      Mitochondrial isolation and characterization

      Porcine left ventricle was homogenized and separated via differential centrifugation, described in supplemental methods. Final mitochondria pellet was resuspended in 300 mM Trehalose, 10 mM HEPES, 10 mM KCl, 1 mM EGTA, 0.1% BSA.
      Antibodies against calnexin (Abcam ab75801), catalase (Proteintech 21260-1-AP), LC3 A/B (CST 12741), Lamin B1 (Proteintech 12987-1-AP), Cox IV (CST 4850), cleaved PARP (BD 564129) and pMLKL (Creative Biolabs PTM-CBMAB-0097LY) were assessed via flow cytometry. Antibodies against calnexin (Abcam ab75801), fibrillarin (Abcam ab226178), α-Tubulin (CST 2125S), and Cox IV (CST 4850S) were assessed via Western blot. BD TruCount, MitoProbe™ JC-1 (ThermoFisher M34152), SYTOX™ (ThermoFisher S10274), IFNγ (RayBiotech ELP-IFNg-1), IL-6 (RayBiotech ELP-IL6-1), GSH/GSSG (Promega V6612), 4-HNE (LSBio LS-C775486-100) 8-OHdG (Rockland 200-301-A99), Annexin V, 7AAD (BioLegend 640922) were assessed using manufacturer's protocols.
      TEM was performed by the Chapel Hill Analytical and Nanofabrication Laboratory. Mitochondrial damage was scored as described.
      • Vincent AE
      • White K
      • Davey T
      • et al.
      Quantitative 3D mapping of the human skeletal muscle mitochondrial network.

      EVLP, cell culture and lung homogenate methods

      EVLP methods were consistent with Toronto-style EVLP,
      • Cypel M
      • Yeung JC
      • Hirayama S
      • et al.
      Technique for prolonged normothermic ex vivo lung perfusion.
      supplemental figure 1. Ex vivo and in vitro methods described in supplemental methods.

      Statistics

      Values are expressed as mean ± SEM analyzed using unpaired, 2-tailed Student's t-test, Mann-Whitney U Test, or one- or 2-way ANOVA with Tukey's multiple comparisons test.

      Results

      Frozen mitochondria demonstrate functional characteristics comparable to fresh isolates.
      To characterize stored mitochondrial isolates, freshly isolated and frozen preparations were evaluated. TEM imaging revealed round structures with densely packed cristae. Freshly isolated preparations (Figure 1A, top) were visually indistinct from preparations stored at -80°C and were largely free of debris (Figure 1A, bottom). Likewise, mitochondrial isolates were devoid of contaminating organelles, as measured by flow cytometry (Figure 1B) and Western blot (Figure 1C). Using TEM imaging, mitochondrial isolates were found to consist of approximately 70% densely packed, class I vesicles, regardless of storage temperature (Figure 1D). Average vesicle size was calculated as 0.97 µm2 via TEM analysis (Figure 1E). These observations were reinforced using flow cytometry, wherein vesicle size remained consistent across 31 frozen preparations (Figure 1F). To demonstrate the significance of storage temperature on maintenance of mitochondria function, fresh isolates were compared with those stored at 4°C or -80°C over 12 months. The mitochondrial coupling state was evaluated using respiratory control ratio (RCR) and the uncoupled, or maximal RCR (RCR-max). Mitochondrial isolates stored at -80°C retained equivalent RCR and RCR-max compared to fresh isolates, while storage at 4°C caused a rapid decline (Figure 1G). Moreover, the number of mitochondrial membrane invaginations (cristae), indicative of healthy mitochondria,
      • Vincent AE
      • White K
      • Davey T
      • et al.
      Quantitative 3D mapping of the human skeletal muscle mitochondrial network.
      remained elevated in fresh and -80°C stored isolates, but not in preparations stored at 4°C (Figure 1H). Mitochondrial swelling, followed by membrane potential disruption and permeability, is a known sequence in mitochondrial damage, inducing cell death.
      • Scarlett JL
      • Murphy MP
      Release of apoptogenic proteins from the mitochondrial intermembrane space during the mitochondrial permeability transition.
      Membrane potential diminished rapidly in isolates stored at 4°C but remained equivalent to fresh isolates when stored at -80°C (Figure 1H). Further, while 4°C storage rapidly increased swelling and permeability, -80°C storage maintained low mitochondrial swelling and permeability, equivalent to fresh preparations (Figure 1I). Lastly, particle count from multiple mitochondrial isolations measured by flow cytometry (Figure 1J) was found to correlate to protein content (Figure 1K). Consequently, particle count was used to establish mitochondrial dosing in subsequent studies utilizing -80°C stored mitochondria.
      Figure 1
      Figure 1Frozen mitochondria demonstrate functional characteristics comparable to fresh isolates. (A) TEM images of mitochondria pellet freshly isolated (top) and following storage at -80°C (bottom). Purity of mitochondrial isolations by flow cytometry (n=3) (B) or Western blot (C). Organelles measured: nuclear (Lamin B1, fibrillarin), endoplasmic reticulum (calnexin), autophagosome (LC3), cytoskeleton (α-tubulin), peroxisome (catalase), mitochondria (COX IV). (D) TEM analysis of mitochondrial damage stratifications immediately after tissue isolation (fresh) or after storage at -80°C (24 hours or 1 week). (E) Mitochondrial size from fresh and frozen preparations calculated by TEM or (F) in frozen preparations by flow cytometry. (G) Impact of storage conditions on mitochondrial RCR and RCR-Max over time (1-6 months storage, n > 10; 7-12 months storage, n = 3). (H) Impact of storage conditions on mitochondrial complexity and membrane potential over time (1-6 months storage, n > 10; 7-12 months storage, n = 3). (I) Impact of storage conditions on mitochondrial swelling and membrane permeability over time (1-6 months storage, n>10; 7-12 months storage, n = 3). Mitochondria particle counts (J) correlated to protein content (Pearson correlation; K). LC3, microtubule-associated protein light chain 3, COX IV, cytochrome c oxidase subunit 4; CM, condensed matrix; RCR, respiratory control ratio; RCR-Max, uncoupled or maximal RCR.

      Mitochondrial transplant reduces lung tissue damage during ex-vivo lung perfusion

      We next sought to evaluate the translational relevance of mitochondrial transplant in a porcine model of EVLP. Lung tissue was exposed to 22 hours of cold ischemia followed by 6 hours of EVLP. Cold ischemia yielded progressively elevated pulmonary vascular resistance (PVR) and reduced oxygenation during EVLP (Figure 2A–B), reflective of known clinical manifestations of PGD.
      • Forgie KA
      • Fialka N
      • Freed DH
      • Nagendran J
      Lung transplantation, pulmonary endothelial inflammation, and ex-situ lung perfusion: a review.
      Mitochondrial infusion, given immediately following assessments at hours 1 and 4, reduced PVR and trended to increase gas exchange (pO2/FiO2) compared to vehicle control (Figure 2 AB). This treatment effect was not due to acute vasodilation, as exogenous mitochondria failed to dilate isolated arteries under control or oxidative conditions (Supplemental Figure 2). Mitochondrial uptake was confirmed in lung endothelial, epithelial, stromal, and immune cell populations using fluorescently labeled mitochondria and histology or flow cytometry evaluation (Supplemental Figure 3). Thus, the impact of mitochondrial transplant on cellular health was examined. RNA sequencing of lung tissue samples taken after 6 hours of EVLP revealed a strong anti-inflammatory signature in mitochondria-treated lungs compared to vehicle control (Figure 2C).
      Figure 2
      Figure 2Mitochondrial transplant reduces lung tissue damage during ex-vivo lung perfusion. Porcine EVLP (A) PVR and (B) gas exchange after 22 hours CIT. Mitochondria were injected at 1 and 4 hours of EVLP (arrows). #p ≤ 0.1 control vs mitochondria (n = 6 control; n = 5 mitochondria) via Mann-Whitney U test; *p ≤ 0.05 control vs mitochondria via Mann-Whitney U test. (C) mRNA signaling pathway analysis on tissue lysate following EVLP. (D) Secreted cytokine analysis from porcine lung tissue homogenate after a 22-hour cold ischemia followed by 4 hours of rewarming. (E) 5-oxoproline and glutamate (one-way ANOVA with Tukey's multiple comparisons test), (F) total and oxidized glutathione (one-way ANOVA with Tukey's multiple comparisons test), (G) Reactive oxygen species byproducts (one-way ANOVA with Tukey's multiple comparisons test), (H) vitamins B1 and B5 (one-way ANOVA with Tukey's multiple comparisons test), (I) viability outcomes (one-way ANOVA with Tukey's multiple comparisons test), and (J) necrosis signaling proteins in porcine lung tissue homogenates after a 22-hour cold ischemia followed by 4 hours of rewarming (one-way ANOVA with Tukey's multiple comparisons test). ^p ≤ 0.05 5-oxoprolone control vs mitochondria (n = 6); *p ≤ 0.05 GSH control vs mitochondria (n = 6); $p ≤ 0.05 GSSG control vs mitochondria (n = 6); %p ≤ 0.05 4-HNE control vs mitochondria (n = 6); a, p ≤ 0.05 8-OHdG control vs mitochondria (n = 6); b, p ≤ 0.05 vitamin B1 control vs mitochondria (n = 6); c, p ≤ 0.05 vitamin B5 control vs mitochondria (n = 6); d, p ≤ 0.05 live control vs mitochondria (n = 6), e, p ≤ 0.05 late apoptotic/necrotic control vs mitochondria (n = 6), f, p ≤ 0.05 pMLKL control vs mitochondria (n = 6), g, p ≤ 0.05 PARP control vs mitochondria (n = 6). PVR, pulmonary vascular resistance; IL-8, interleukin-8; IL-6, interleukin-6; STAT3, signal transducer and activator of transcription 3; IL-1, Interleukin-1; mTOR, mammalian target of rapamycin; eNOS, endothelial nitric oxide synthase; TNFR, tumor necrosis factor receptor; IFN, interferon; NF-kB, nuclear factor kappa B; IFNg, interferon gamma; GSH, total glutathione; GSSG, oxidized glutathione; 4-HNE, 4-hydroxynonenal; 8-OHdG, 8-Oxo-2′-deoxyguanosine; pMLKL, phospho-mixed lineage kinase domain-like protein; PARP, poly (ADP-ribose) polymerase.
      To model cellular behavior during EVLP, a lung tissue homogenate system was established. Porcine lung was processed into a single cell suspension and placed in a refrigerated incubator for 22 hours followed by 4 hours of rewarming. Like EVLP tissue data (Figure 2C), mitochondrial transplant given upon rewarming reduced homogenate INFg and IL-6 secretions in a dose-responsive manner compared to vehicle control (Figure 2D). Further, metabolomics analysis revealed a dose-responsive elevation in 5-oxoproline and glutamate after mitochondrial transplant, indicative of glutathione synthesis (
      • Van Der Werf P
      • Stephani RA
      • Meister A
      Accumulation of 5-oxoproline in mouse tissues after inhibition of 5-oxoprolinase and administration of amino acids: evidence for function of the gamma-glutamyl cycle.
      ; Figure 2E). In support of this hypothesis, total glutathione (GSH) was increased but oxidized glutathione (GSSG) was reduced (Figure 2F) after mitochondrial transplant. Further, oxidative by-products 4-HNE (lipid peroxidation) and 8-OHdG (DNA oxidation) were reduced to less than half that of IRI control after mitochondrial transplant at 1000 particles/mg (Figure 2G). Likewise, mitochondrial transplant increased vitamin B1, an antioxidant and co-factor in amino acid and glucose metabolism, and vitamin B5, a critical component of coenzyme A, in a dose-responsive manner,
      • Depeint F
      • Bruce WR
      • Shangari N
      • Mehta R
      • O'Brien PJ
      Mitochondrial function and toxicity: role of the B vitamin family on mitochondrial energy metabolism.
      (Figure 2H). Ischemia followed by rewarming in lung tissue homogenate resulted in 40% of the cell population undergoing late stage apoptotic or necrotic death as measured by nucleic acid staining (Figure 2I). Mitochondrial transplant during rewarming reduced cell death by over 30% and increased live cell count from 30% to 40%, a 25% boost in viability (Figure 2I). To further explore this cellular salvage, apoptotic and necrotic signaling were evaluated. A mediator of apoptotic cell death, poly (ADP-ribose) polymerase (PARP) cleavage is now understood to play a role in both apoptotic and necrotic death.
      • Boulares AH
      • Yakovlev AG
      • Ivanova V
      • et al.
      Role of poly(ADP-ribose) polymerase (PARP) cleavage in apoptosis. Caspase 3-resistant PARP mutant increases rates of apoptosis in transfected cells.
      ,
      • Ha HC
      • Snyder SH
      Poly(ADP-ribose) polymerase is a mediator of necrotic cell death by ATP depletion.
      Mitochondrial treatment moderately reduced cleaved PARP at 500 and 1000 particles/mg tissue compared to vehicle control (Figure 2J). Phosphorylated MLKL, an established driver of necrotic membrane permeability, was dramatically reduced by 40% and 65% compared to vehicle when treated at 500 or 1000 particles/mg tissue, respectively,
      • Wang H
      • Sun L
      • Su L
      • et al.
      Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3.
      (Figure 2J). These data are indicative of mitochondrial-mediated improvement in necrotic cellular death following IRI.

      Mitochondrial transplant improves lung function and reduces inflammatory signaling in human EVLP

      To substantiate therapeutic potential, the impact of mitochondria given during and after human EVLP was evaluated. Bilateral human lungs rejected for clinical transplantation (Supplemental Table 1) were assessed for 4 hours, cooled, and stored on ice for 18 hours to mimic an extended shipment of lung tissue from the EVLP site to the clinic. Mitochondria or vehicle control was added during perfusion immediately following the second hour of EVLP and at the EVLP conclusion, such that mitochondria or vehicle control was present during hours 3 and 4 of EVLP and during the second cold ischemic time. Unique from porcine dosing, mitochondrial transplant was given at hour 2 in human EVLP to extend pre-dosing evaluation, anticipating the need to control for heightened donor variability in the human model. Like porcine EVLP, mitochondrial transplant reduced PVR (Figure 3A) and increased gas exchange (Figure 3B) in human lungs compared to vehicle control. In support of recent studies, no oxidative or proinflammatory response was observed following injection of a porcine-derived mitochondrial transplant in human tissue.
      • Ramirez-Barbieri G
      • Moskowitzova K
      • Shin B
      • et al.
      Alloreactivity and allorecognition of syngeneic and allogeneic mitochondria.
      • Pourmohammadi-Bejarpasi Z
      • Roushandeh AM
      • Saberi A
      • et al.
      Mesenchymal stem cells-derived mitochondria transplantation mitigates I/R-induced injury, abolishes I/R-induced apoptosis, and restores motor function in acute ischemia stroke rat model.
      • Huang PJ
      • Kuo CC
      • Lee HC
      • et al.
      Transferring xenogenic mitochondria provides neural protection against ischemic stress in ischemic rat brains.
      In fact, mitochondrial transplant reduced the ROS byproduct 8-OHdG as evaluated via immunohistochemistry (Figure 3C). To confirm mitochondrial uptake and retention in human tissue, porcine-specific mitochondrial ND5 (SsMtND5) mRNA was evaluated. Transplanted porcine mitochondria were present in all lobes, with the highest expression detected in the upper region of the right inferior lobe (Figure 3D). To interrogate the cellular impact of mitochondrial uptake, tissue from the right inferior proximal region was evaluated for proinflammatory cytokines, ROS, and soluble adhesion molecules, which have been associated with poor transplant outcome.
      • Andreasson AS
      • Karamanou DM
      • Gillespie CS
      • et al.
      Profiling inflammation and tissue injury markers in perfusate and bronchoalveolar lavage fluid during human ex vivo lung perfusion.
      • Machuca TN
      • Cypel M
      • Yeung JC
      • et al.
      Protein expression profiling predicts graft performance in clinical ex vivo lung perfusion.
      • Hashimoto K
      • Cypel M
      • Kim H
      • et al.
      Soluble adhesion molecules during ex vivo lung perfusion are associated with posttransplant primary graft dysfunction.
      Mitochondrial infusion reduced circulating proinflammatory cytokines CXCL8 and CXCL10 during EVLP (Figure 3E). Further, exogenous mitochondria decreased mRNA levels of IL1β, IL-6, CXCL8, CCL2 and CXCL10 compared to vehicle control (Figure 3F) and reduced proinflammatory cytokine proteins in lung tissue lysate following EVLP (Figure 3G). Similarly, soluble adhesion molecule VCAM1 was reduced during mitochondrial-treated EVLP compared to control (Figure 3H). Further, ICAM1, CD44, and VCAM1 activation-associated adhesion molecule mRNA (Figure 3I) and protein (Figure 3J) levels were reduced following mitochondrial transplant. These results were reinforced in human pulmonary artery endothelial cells under oxidative stress, wherein exogenous frozen mitochondria reduced pro-inflammatory cytokine secretion, but mitochondria stored at 4°C did not (Supplemental Figure 4). Further, intact mitochondria were required for this function, as whole organelle but not mitochondrial-derived proteins or vesicles were responsible for the anti-inflammatory effect (Supplemental Figure 5). Consistent with porcine tissue analysis (Figure 2), exogenous mitochondrial transplant improved human lung function, acting to reduce cellular inflammation and oxidative damage.
      Figure 3
      Figure 3Mitochondrial treatment improves lung function and reduces inflammatory signaling in human EVLP. EVLP (A) PVR (Mann-Whitney U test) and (B) PO2/FiO2 (Mann-Whitney U test). Mitochondria isolates were injected at 2 hours of EVLP (arrows). (C) Tissue 8′OHdG via immunohistochemistry taken from the proximal region of the right inferior lobe (images) and analyzed from regions sampled from all lobes (quantification; unpaired Student's t-test). (D) Porcine mitochondrial uptake evaluated by mRNA expression of ssMtND5 and assessed in different regions of the lung. Cytokines evaluated in (E) EVLP circulating perfusate (2-way ANOVA with Tukey's multiple comparisons test), (F) tissue lysate mRNA (unpaired Student's t-test) and (G) tissue lysate protein (unpaired Student's t-test). Adhesion molecules evaluated in (H) EVLP circulating perfusate (2-way ANOVA with Tukey's multiple comparisons test), (I) tissue lysate mRNA (unpaired Student's t-test) and (J) tissue lysate protein (unpaired Student's t-test). *p ≤ 0.05 control vs mitochondria (control n = 6, mitochondria n = 5); #p ≤ 0.1 control vs mitochondria (control n = 6, mitochondria n = 5). Scale bar (C) represents 100 µM. PVR, pulmonary vascular resistance; 8-OHdG, 8-Oxo-2′-deoxyguanosine; SsMtND5, porcine NADH-ubiquinone oxidoreductase chain 5 protein; IL-1B, interleukin-1b; IL86, interleukin-6; IL-8, interleukin-8, CCL2, C-C motif chemokine ligand 2; IL-10, interleukin-10; ICAM1, intercellular adhesion molecule 1; VCAM1, vascular cell adhesion protein 1.

      Mitochondrial transplant functions through autophagy

      A recycling system for cells, autophagy improves downstream cellular inflammation, oxidative stress, and necrosis by removing damaged organelles for use in energy production.
      • Kalogeris T
      • Baines CP
      • Krenz M
      • Korthuis RJ
      Ischemia/Reperfusion.
      ,
      • Ornatowski W
      • Lu Q
      • Yegambaram M
      • et al.
      Complex interplay between autophagy and oxidative stress in the development of pulmonary disease.
      To examine the possibility that mitochondrial transplant promotes cellular salvage through autophagy, relevant gene signatures were probed in human lung tissue after 4 hours of EVLP. TFEB, a master autophagy regulator, and autophagy induction complex components Beclin-1 and ULK1 were elevated following mitochondrial transplant during EVLP compared to vehicle control. (Figure 4A). We next evaluated direct activation of autophagy by mitochondrial transplant using simulated IRI in an autophagy reporter cell line using 22-24-hours of hypothermia exposure followed by 4 hours of rewarming. In this system, the LC3 reporter tag is degraded during autophagy, thereby diminishing signal. Exogenous mitochondrial transplant resulted in LC3-reporter degradation in a dose dependent manner, independent of cellular viability, which was maintained, supportive of activated autophagy (Figure 4B). To identify the specific pathway by which mitochondrial transplant promotes autophagy, several targeted inhibitors were examined. Autophagy initiation and phagophore formation were addressed using the ATG4 inhibitor NSC185058.
      • Akin D
      • Wang SK
      • Habibzadegah-Tari P
      • et al.
      A novel ATG4B antagonist inhibits autophagy and has a negative impact on osteosarcoma tumors.
      Nucleation was targeted using the MEK inhibitor U-0126 and the PI3K inhibitors wortmannin and LY294002.
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      • Chow S
      • Shankey TV
      Cytometry of intracellular signaling: from laboratory bench to clinical application.
      ,
      • Blommaart EF
      • Krause U
      • Schellens JP
      • Vreeling-Sindelarova H
      • Meijer AJ
      The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes.
      Finally, bafilomycin A1 was used as a late-stage autophagy inhibitor of autophagosome fusion with lysosomes.
      • Mauvezin C
      • Neufeld TP
      Bafilomycin A1 disrupts autophagic flux by inhibiting both V-ATPase-dependent acidification and Ca-P60A/SERCA-dependent autophagosome-lysosome fusion.
      Using the autophagy reporter cell line under simulated IRI conditions and mitochondrial transplant at 800 particles/cell, ATG4 inhibition had minimal impact on the mitochondrial induction of autophagy. PI3K and late-stage autophagy inhibition trended to blunt mitochondrial-induced autophagy while treatment with the MEK inhibitor U-0126 gave the most dramatic response. Indeed, U-0126 partially reversed the mitochondrial-mediated activation in autophagy, blunting the degradation of LC3 by 41% (Figure 4C). Thus, we propose that 1 specific protein target of mitochondrial transplant is MEK, activation of which subsequently promotes late-stage autophagy. Supporting this hypothesis, mitochondrial transplant induced autophagy as measured by dye incorporation into autophagosome and autolysosomes in the lung homogenate model of IRI described in figure 2D (Figure 4D). Addition of the MEK inhibitor U-0126 prevented autophagy induction at a dose of 500 particles/mg and partially prevented at 1000 particles/mg tissue (Figure 4D), indicating that higher mitochondrial doses overcome U-0126 inhibition through direct competition or through stimulation of non-MEK signaling. To evaluate the impact of autophagy on proposed downstream pathways, cytokine secretion was measured from HPAECs exposed to simulated IRI. As expected, exogenous mitochondria reduced secretion of pro-inflammatory CXCL8, MCP-1 and GROα in a dose-responsive manner compared to vehicle control. Pre-treatment with U-0126 partially reversed the effect of mitochondrial transplant (Figure 4E). Similarly, mitochondrial transplant reduced 4-HNE and 8-OHdG, an effect which was partly prevented by U-0126 (Figure 4F). Further, mitochondrial transplant increased cellular viability compared to ischemia rewarming injury, a treatment effect which was partially prevented by MEK inhibition (Figure 4G). Likewise, while mitochondrial transplant reduced phosphorylated MLKL compared to vehicle, U-0126 partially reversed this marker of tissue necrosis (Figure 4H). Finally, total ATP in HPAECs was measured following ischemia and rewarming injury. Mitochondrial transplant increased the available ATP pool in a dose-responsive manner, which was fully reversed by U-0126 under 100 particles/cell, and partially reversed under 1000 particles/cell (Figure 4I). Importantly, therapeutic benefits from a single dose of exogenous mitochondria were maintained for several days in HPAEC following ischemia and rewarming injury (Supplemental Figure 6). These data support the hypothesis that exogenous mitochondria upregulate autophagy and thus decrease downstream inflammation, reactive oxygen species, and necrosis in IRI.
      Figure 4
      Figure 4Mitochondrial transplant functions through autophagy. Gene expression of tissue autophagy signaling (unpaired Student's t-test; A). The autophagy reporter cell line LC3 HiBiT with increasing doses of exogenous mitochondria (B) or under 800 mitochondria particles/cell after exposure to targeted autophagy pathway inhibitors (one-way ANOVA with Tukey's multiple comparisons test; C). (D) Autophagy induction in porcine lung homogenates exposed to 24 hours at 4 and 4 hours rewarming and mitochondria at 500 or 1000 particles/mg, with or without the addition of the autophagy inhibitor U-0126 (one-way ANOVA with Tukey's multiple comparisons test). HPAECs exposed to 24 hours at 4 and 4 hours of rewarming were pre-treated with U-0126 and 500 or 1000 particles/cell mitochondria and assessed for (E) cytokine secretion, (F) 4-HNE and 8-Ohdg (one-way ANOVA with Tukey's multiple comparisons test), (G) viability (one-way ANOVA with Tukey's multiple comparisons test), (H) necrosis-associated protein pMLKL (one-way ANOVA with Tukey's multiple comparisons test) and (I) ATP production (one-way ANOVA with Tukey's multiple comparisons test). a, p ≤ 0.05 control vs mitochondria (control n = 6; mitochondria n = 4); %P ≤ 0.05 control vs mitochondria 1000 particles (n = 3); $p ≤ 0.05 mitochondria vs mitochondria + inhibitor (n = 3); *p ≤ 0.05 IRI control vs mitochondria (lung homogenate n = 6; HPAEC n = 3); ^p ≤ 0.05 Low dose mitochondria (500 or 100) particles vs mitochondria (500 or 100) particles + U-0126 (lung homogenate n = 6; HPAEC n = 3); #p ≤ 0.05 Mitochondria vs mitochondria + U-0126 (n = 3). CXCL8, interleukin-8; MCP-1, monocyte chemoattractant protein-1; GROa, growth regulated alpha protein; 4-HNE, 4-hydroxynonenal; 8-OHdG, 8-Oxo-2′-deoxyguanosine; pMLKL, phospho-mixed lineage kinase domain-like protein; ATP, adenosine triphosphate.

      Proposed mitochondrial mechanism of action

      Data herein support the idea that mitochondrial transplant promotes autophagy through upregulation of the MEK nucleation pathway. Promotion of autophagy during lung IRI subsequently reduces pro-inflammatory cytokine signaling and secretion, reduces by-products of ROS, and boosts antioxidant signaling. By recycling damaged cells, autophagy also acts to provide metabolites for anaplerosis and thus bolsters the ATP pool.
      • Lahiri V
      • Hawkins WD
      • Klionsky DJ
      Watch what you (self-) eat: autophagic mechanisms that modulate metabolism.
      Together, these protective benefits result in cellular salvage and protection from IRI-induced tissue injury (Figure 5).
      Figure 5
      Figure 5Proposed mitochondria mechanism of action. We propose that mitochondrial transplant activates MEK during IRI, thus promoting autophagy. Autophagy curbs downstream cytokine signaling and secretion (anti-inflammatory), reduces reactive oxygen species (ROS) by-products and increases antioxidant signaling (ROS reduction), and provides metabolites for ATP generation via anaplerosis (energy homeostasis), resulting in cellular salvage (pro survival). AO, antioxidant; ATP, adenosine triphosphate; TCA, tricarboxylic acid cycle; ROS, reactive oxygen species.

      Discussion

      Our findings provide evidence of the beneficial effect of mitochondrial isolates on cellular damage after injurious ischemia reperfusion. We propose that mitochondria isolated from porcine heart tissue can be stored and used as an allogenic xenograft therapy during ex-vivo lung perfusion (EVLP). Compellingly, data herein suggest that mitochondrial transplant during EVLP may reduce the risk of PGD in lung transplant recipients. This proposal is congruent with early work identifying ischemia-reperfusion tissue injury upstream of acute organ rejection
      • Land W
      The potential impact of the reperfusion injury on acute and chronic rejection events following organ transplantation.
      ,
      • Land W
      Postischemic reperfusion injury and allograft dysfunction: is allograft rejection the result of a fateful confusion by the immune system of danger and benefit?.
      and the finding that interventions targeting IRI resulted in decreased incidence of acute graft rejection.
      • Land W
      • Schneeberger H
      • Schleibner S
      • et al.
      The beneficial effect of human recombinant superoxide dismutase on acute and chronic rejection events in recipients of cadaveric renal transplants.
      Mitochondrial transplant is one such intervention, with demonstrated improvement in mouse lung function when given via in vivo infusion following IRI.
      • Moskowitzova K
      • Shin B
      • Liu K
      • et al.
      Mitochondrial transplantation prolongs cold ischemia time in murine heart transplantation.
      Indeed, a systemic review evaluating animal and human studies to date support the potential benefit of mitochondrial transplant in the treatment of IRI.
      • Hayashida K
      • Takegawa R
      • Shoaib M
      • et al.
      Mitochondrial transplantation therapy for ischemia reperfusion injury: a systematic review of animal and human studies.
      Building on these studies, mitochondrial transplant herein reduced tissue inflammation and reactive oxygen species (ROS) accumulation after IRI (Figure 2C–G), resulting in cellular salvage and improved lung function (Figure 2A–B, I–J Figure 3A–B).
      While the nonclinical benefits of mitochondrial isolates in cardiac IRI have been demonstrated and clinical trials are promising,
      • Guariento A
      • Piekarski BL
      • Doulamis IP
      • et al.
      Autologous mitochondrial transplantation for cardiogenic shock in pediatric patients following ischemia-reperfusion injury.
      doubt of their therapeutic utility remains.
      • Bertero E
      • Maack C
      • O'Rourke B
      Mitochondrial transplantation in humans: “magical” cure or cause for concern?.
      Bertero and colleagues bring up several important concerns, questioning the ability of intact mitochondria to enter a calcium rich extracellular environment and retain cellular uptake and functionality. One possible explanation could be a paracrine effect, wherein mitochondria-derived vesicles could account for some of the benefit of mitochondrial transplant.
      • Sugiura A
      • McLelland GL
      • Fon EA
      • McBride HM
      A new pathway for mitochondrial quality control: mitochondrial-derived vesicles.
      To examine this possibility, published inhibitors of exogenous mitochondrial uptake could be utilized to gain insight.
      • Pacak CA
      • Preble JM
      • Kondo H
      • et al.
      Actin-dependent mitochondrial internalization in cardiomyocytes: evidence for rescue of mitochondrial function.
      ,
      • Kesner EE
      • Saada-Reich A
      • Lorberboum-Galski H
      Characteristics of mitochondrial transformation into human cells.
      However many mechanisms of uptake have been confirmed, including exocytosis and endocytosis of free mitochondria, cytoplasmic fusion and gap junction formation, formation of tunneling nanotubes, and secretion of extracellular vesicles.
      • Qin Y
      • Jiang X
      • Yang Q
      • Zhao J
      • Zhou Q
      • Zhou Y
      The functions, methods, and mobility of mitochondrial transfer between cells.
      Therefore, inhibition of any specific uptake pathway may not address redundant mechanisms. Thus, to identify any contribution of mitochondria-derived paracrine signaling, intact mitochondria were isolated from mitochondria-derived proteins or vesicles via centrifugation. Data herein suggest that uptake of intact mitochondria, and not organelle secretions are responsible for therapeutic activity (Supplemental Figures 5A-D). Moreover, intact, functional mitochondria were required for performance, suggesting that mitochondrial-derived protein and genetic material outside of intact organelles was not sufficient for activity (Supplemental Figure 3). While the mechanism by which intact mitochondria survive extracellular calcium fluctuations remains to be determined, evidence of this phenomenon has become increasingly recognized. Healthy cells are known to transfer mitochondria, thus restoring the biological fitness of damaged recipient cells.
      • Valenti D
      • Vacca RA
      • Moro L
      • Atlante A
      Mitochondria can cross cell boundaries: an overview of the biological relevance, pathophysiological implications and therapeutic perspectives of intercellular mitochondrial transfer.
      Here, we sought to harness this cellular altruism and demonstrate its potential use as an off-the-shelf therapeutic.
      The ability to store functional mitochondria is a controversial concept. In fact, autologous mitochondria were found to be significantly less active when stored on ice for more than 1 hour, necessitating rapid preparation of fresh isolates to support clinical treatment.
      • McCully JD
      • Cowan DB
      • Emani SM
      • Del Nido PJ.
      Mitochondrial transplantation: from animal models to clinical use in humans.
      These findings were in line with others who have attempted storage with limited success.
      • Yamada Y
      • Ito M
      • Arai M
      • Hibino M
      • Tsujioka T
      • Harashima H
      Challenges in promoting mitochondrial transplantation therapy.
      Because the use of mitochondrial isolates to treat IRI is promising,
      • Hayashida K
      • Takegawa R
      • Shoaib M
      • et al.
      Mitochondrial transplantation therapy for ischemia reperfusion injury: a systematic review of animal and human studies.
      but fresh isolation limits product utility, the establishment of a method that permits mitochondrial storage is critical. Herein, we demonstrate the ability to store mitochondrial isolates using a Trehalose-containing buffer at -80°C to maximize protein stability. Critically, this storage protocol maintained mitochondrial health and functionality for up to 1 year (Figure 1G–I) and retained functional benefit in vitro and in EVLP (Figure 2, Figure 3, Figure 4). EVLP, chosen as a controlled and sensitive evaluation of lung function, would be well supported by a lung transplant model in future studies. Importantly, despite the rapid decline in exogenous porcine mitochondrial mRNA in HPAECs exposed to cold ischemia and then rewarming, the antioxidant and anti-inflammatory benefits of a single dose remained for 7 days (Supplemental Figure 6F). These data suggest that exogenous mitochondria boost cellular health via host cell remodeling. Therefore, a single dose of mitochondria may be sufficient to impact sustained benefit following IRI. In summary, data herein suggest mitochondrial transplant functions to improve IRI-associated mitochondrial dysfunction, providing potential benefit for patients with PGD.

      Authors' contributions

      CC participated in study design, data analysis, writing. CG participated in data acquisition, writing. YTL, LB, LR, ZL participated in data acquisition and analysis. SP, RS, JZ, TZ, BJ participated in data acquisition. SH participated in study design, data acquisition, analysis, writing. TP participated in study design, data interpretation.

      Disclosure statement

      Authors are paid employees of United Therapeutics. No author has or will receive financial incentive of any kind in relation to the technology described herein. United Therapeutics affiliation does not alter adherence to JHLT policies on sharing data and materials. The authors declare an associated nonprovisional patent (WO2020257281A1) covering the use of stored mitochondria in the treatment of transplant-associated lung tissue.

      Acknowledgments

      The authors thank Kimwa Walker for her work embedding and cutting samples for immunohistochemistry. We thank the Regenerative Medicine Laboratory members at United Therapeutics for support in this study.

      Appendix. Supplementary materials

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