Expanding Donor Options for Lung Transplant: Extended Criteria, Donation After Circulatory Death, ABO Incompatibility, and Evolution of Ex Vivo Lung Perfusion.

Only using brain-dead donors with standard criteria, the existing donor shortage has never improved in lung transplantation. Currently, clinical efforts have sought the means to use cohorts of untapped donors, such as extended criteria donors, donation after circulatory death, and donors that are ABO blood group incompatible, and establish the evidence for their potential contribution to the lung transplant needs. Also, technical maturation for using those lungs may eliminate immediate concerns about the early posttransplant course, such as primary graft dysfunction or hyperacute rejection. In addition, recent clinical and preclinical advances in ex vivo lung perfusion techniques have allowed the safer use of lungs from high-risk donors and graft modification to match grafts to recipients and may improve posttransplant outcomes. This review summarizes recent trends and accomplishments and future applications for expanding the donor pool in lung transplantation.

Cross-Regulation of F-Box Protein FBXL2 with T-bet and TNF-α during Acute and Chronic Lung Allograft Rejection.

Chronic lung allograft dysfunction is the major barrier to long-term survival in lung transplant recipients. Evidence supports type 1 alloimmunity as the predominant response in acute/chronic lung rejection, but the immunoregulatory mechanisms remain incompletely understood. We studied the combinatorial F-box E3 ligase system: F-box protein 3 (FBXO3; proinflammatory) and F-box and leucine-rich repeat protein 2 (FBXL2; anti-inflammatory and regulates TNFR-associated factor [TRAF] protein). Using the mouse orthotopic lung transplant model, we evaluated allografts from BALB/c → C57BL/6 (acute rejection; day 10) and found significant induction of FBXO3 and diminished FBXL2 protein along with elevated T-bet, IFN-γ, and TRAF proteins 1-5 compared with isografts. In the acute model, treatment with costimulation blockade (MR1/CTLA4-Ig) resulted in attenuated FBXO3, preserved FBXL2, and substantially reduced T-bet, IFN-γ, and TRAFs 1-5, consistent with a key role for type 1 alloimmunity. Immunohistochemistry revealed significant changes in the FBXO3/FBXL2 balance in airway epithelia and infiltrating mononuclear cells during rejection compared with isografts or costimulation blockade-treated allografts. In the chronic lung rejection model, DBA/2J/C57BL/6F1 > DBA/2J (day 28), we observed persistently elevated FBXO3/FBXL2 balance and T-bet/IFN-γ protein and similar findings from lung transplant recipient lungs with chronic lung allograft dysfunction versus controls. We hypothesized that FBXL2 regulated T-bet and found FBXL2 was sufficient to polyubiquitinate T-bet and coimmunoprecipitated with T-bet on pulldown experiments and vice versa in Jurkat cells. Transfection with FBXL2 diminished T-bet protein in a dose-dependent manner in mouse lung epithelial cells. In testing type 1 cytokines, TNF-α was found to negatively regulate FBXL2 protein and mRNA levels. Together, our findings show the combinatorial E3 ligase FBXO3/FBXL2 system plays a role in the regulation of T-bet through FBXL2, with negative cross-regulation of TNF-α on FBXL2 during lung allograft rejection.

Metabolic Syndrome Mediates ROS-miR-193b-NFYA-Dependent Downregulation of Soluble Guanylate Cyclase and Contributes to Exercise-Induced Pulmonary Hypertension in Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Many patients with heart failure with preserved ejection fraction have metabolic syndrome and develop exercise-induced pulmonary hypertension (EIPH). Increases in pulmonary vascular resistance in patients with heart failure with preserved ejection fraction portend a poor prognosis; this phenotype is referred to as combined precapillary and postcapillary pulmonary hypertension (CpcPH). Therapeutic trials for EIPH and CpcPH have been disappointing, suggesting the need for strategies that target upstream mechanisms of disease. This work reports novel rat EIPH models and mechanisms of pulmonary vascular dysfunction centered around the transcriptional repression of the soluble guanylate cyclase (sGC) enzyme in pulmonary artery (PA) smooth muscle cells. METHODS: We used obese ZSF-1 leptin-receptor knockout rats (heart failure with preserved ejection fraction model), obese ZSF-1 rats treated with SU5416 to stimulate resting pulmonary hypertension (obese+sugen, CpcPH model), and lean ZSF-1 rats (controls). Right and left ventricular hemodynamics were evaluated using implanted catheters during treadmill exercise. PA function was evaluated with magnetic resonance imaging and myography. Overexpression of nuclear factor Y α subunit (NFYA), a transcriptional enhancer of sGC ß1 subunit (sGCß1), was performed by PA delivery of adeno-associated virus 6. Treatment groups received the SGLT2 inhibitor empagliflozin in drinking water. PA smooth muscle cells from rats and humans were cultured with palmitic acid, glucose, and insulin to induce metabolic stress. RESULTS: Obese rats showed normal resting right ventricular systolic pressures, which significantly increased during exercise, modeling EIPH. Obese+sugen rats showed anatomic PA remodeling and developed elevated right ventricular systolic pressure at rest, which was exacerbated with exercise, modeling CpcPH. Myography and magnetic resonance imaging during dobutamine challenge revealed PA functional impairment of both obese groups. PAs of obese rats produced reactive oxygen species and decreased sGCß1 expression. Mechanistically, cultured PA smooth muscle cells from obese rats and humans with diabetes or treated with palmitic acid, glucose, and insulin showed increased mitochondrial reactive oxygen species, which enhanced miR-193b-dependent RNA degradation of nuclear factor Y α subunit (NFYA), resulting in decreased sGCß1-cGMP signaling. Forced NYFA expression by adeno-associated virus 6 delivery increased sGCß1 levels and improved exercise pulmonary hypertension in obese+sugen rats. Treatment of obese+sugen rats with empagliflozin improved metabolic syndrome, reduced mitochondrial reactive oxygen species and miR-193b levels, restored NFYA/sGC activity, and prevented EIPH. CONCLUSIONS: In heart failure with preserved ejection fraction and CpcPH models, metabolic syndrome contributes to pulmonary vascular dysfunction and EIPH through enhanced reactive oxygen species and miR-193b expression, which downregulates NFYA-dependent sGCß1 expression. Adeno-associated virus-mediated NFYA overexpression and SGLT2 inhibition restore NFYA-sGCß1-cGMP signaling and ameliorate EIPH.

Cyclosporin A Administration During Ex Vivo Lung Perfusion Preserves Lung Grafts in Rat Transplant Model.

BACKGROUND: Despite the benefits of ex vivo lung perfusion (EVLP) such as lung reconditioning, preservation, and evaluation before transplantation, deleterious effects, including activation of proinflammatory cascades and alteration of metabolic profiles have been reported. Although patient outcomes have been favorable, further studies addressing optimal conditions are warranted. In this study, we investigated the role of the immunosuppressant drug cyclosporine A (CyA) in preserving mitochondrial function and subsequently preventing proinflammatory changes in lung grafts during EVLP. METHODS: Using rat heart-lung blocks after 1-hour cold preservation, an acellular normothermic EVLP system was established for 4 hours. CyA was added into perfusate at a final concentration of 1 µM. The evaluation included lung graft function, lung compliance, and pulmonary vascular resistance as well as biochemical marker measurement in the perfusate at multiple time points. After EVLP, single orthotopic lung transplantation was performed, and the grafts were assessed 2 hours after reperfusion. RESULTS: Lung grafts on EVLP with CyA exhibited significantly better functional and physiological parameters as compared with those without CyA treatment. CyA administration attenuated proinflammatory changes and prohibited glucose consumption during EVLP through mitigating mitochondrial dysfunction in lung grafts. CyA-preconditioned lungs showed better posttransplant lung early graft function and less inflammatory events compared with control. CONCLUSIONS: 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.

Impact of triptolide during ex vivo lung perfusion on grafts after transplantation in a rat model.

OBJECTIVE: Ex vivo lung perfusion creates a proinflammatory environment leading to deterioration in graft quality that may contribute to post-transplant graft dysfunction. Triptolide has been shown to have a therapeutic potential in various disease states because of its anti-inflammatory properties. On this basis, we investigated the impact of triptolide on graft preservation during ex vivo lung perfusion and associated post-transplant outcomes in a rat transplant model. METHODS: We performed rat normothermic ex vivo lung perfusion with acellular Steen solution containing 100 nM triptolide for 4 hours and compared the data with untreated lungs. Orthotopic single lung transplantation after ex vivo lung perfusion was performed. RESULTS: Physiologic and functional parameters of lung grafts on ex vivo lung perfusion with triptolide were better than those without treatment. Graft glucose consumption was significantly attenuated on ex vivo lung perfusion with triptolide via inhibition of hypoxia signaling resulting in improved mitochondrial function and reduced oxidative stress. Also, intragraft inflammation was markedly lower in triptolide-treated lungs because of inhibition of nuclear factor-κB signaling. Furthermore, post-transplant graft function and inflammatory events were significantly improved in the triptolide group compared with the untreated group. CONCLUSIONS: Treatment of lung grafts with triptolide during ex vivo lung perfusion may serve to enhance graft preservation and improve graft protection resulting in better post-transplant outcomes.

Bronchial-arterial-circulation-sparing Lung Preservation: A New Organ Protection Approach for Lung Transplantation.

BACKGROUND: Compromised microvasculature resulting from disrupted bronchial arterial circulation appears to trigger chronic lung allograft dysfunction. Maintaining the microvasculature throughout the transplant process could improve the long-term health of transplanted lungs. We recently developed a bronchial-arterial-circulation-sparing (BACS) lung preservation approach and tested whether this approach would decrease microvascular damage and improve allograft function. METHODS: The lungs of Lewis rats were procured using either the BACS approach, where the bronchial and pulmonary arteries were synchronously perfused; a conventional approach, where only the pulmonary artery was perfused; or a conventional approach with a prostaglandin flush. After 4 hours of cold ischemia, physiologic function and vascular tone of the grafts were evaluated during ex vivo lung perfusion (EVLP), and microvasculature damage was assessed using 2-photon microscopy analysis. Lung function was compared after transplant among the groups. RESULTS: After 4 hours of cold ischemia, the BACS group exhibited significantly higher adenosine triphosphate levels and lower expression of phosphorylated myosin light chain, which is essential for vascular smooth muscle contraction. On EVLP, the BACS and prostaglandin groups showed lower pulmonary vascular resistance and less arterial stiffness. BACS attenuated microvasculature damage in the lung grafts when compared with conventional preservation. After transplantation, the lungs preserved with the BACS approach exhibited significantly better graft function and lower expression of phosphorylated myosin light chain. CONCLUSIONS: Our data suggest that BACS lung preservation protects the dual circulation inherent to the lungs, facilitating robust microvasculature in lung grafts after transplantation, leading to better posttransplant outcomes.

The Bronchial Arterial Circulation in Lung Transplantation: Bedside to Bench to Bedside, and Beyond.

Chronic allograft dysfunction (CLAD) remains a major complication, causing the poor survival after lung transplantation (Tx). Although strenuous efforts have been made at preventing CLAD, surgical approaches for lung Tx have not been updated over the last 2 decades. The bronchial artery (BA), which supplies oxygenated blood to the airways and constitutes a functional microvasculature, has occasionally been revascularized during transplants, but this technique did not gain popularity and is not standard in current lung Tx protocols, despite the fact that a small number of studies have shown beneficial effects of BA revascularization on limiting CLAD. Also, recent basic and clinical evidence has demonstrated the relationship between microvasculature damage and CLAD. Thus, the protection of the bronchial circulation and microvasculature in lung grafts may be a key factor to overcome CLAD. This review revisits the history of BA revascularization, discusses the role of the bronchial circulation in lung Tx, and advocates for novel bronchial-arterial-circulation sparing approaches as a future direction for overcoming CLAD. Although there are some already published review articles summarizing the surgical techniques and their possible contribution to outcomes in lung Tx, to the best of our knowledge, this review is the first to elaborate on bronchial circulation that will contribute to prevent CLAD from both scientific and clinical perspectives: from bedside to bench to bedside, and beyond.

Targeting Circulating Leukocytes and Pyroptosis During Ex Vivo Lung Perfusion Improves Lung Preservation.

BACKGROUND: The role of the circulating leukocytes in lungs and their relationship with circulating proinflammatory cytokines during ischemia-reperfusion injury is not well understood. Using ex vivo lung perfusion (EVLP) to investigate the pathophysiology of isolated lungs, we aimed to identify a therapeutic target to optimize lung preservation leading to successful lung transplantation. METHODS: Rat heart-lung blocks were placed on EVLP for 4 hours with or without a leukocyte-depleting filter (LF). After EVLP, lung grafts were transplanted, and posttransplant outcomes were compared. RESULTS: Lung function was significantly better in lung grafts on EVLP with a LF than in lungs on EVLP without a LF. The interleukin (IL)-6 levels in the lung grafts and EVLP perfusate were also significantly lower after EVLP with a LF. Interestingly, IL-6 levels in the perfusate did not increase after the lungs were removed from the EVLP circuit, indicating that the cells trapped by the LF were not secreting IL-6. The trapped cells were analyzed with flow cytometry to detect apoptosis and pyroptosis; 26% were pyroptotic (Caspase-1-positive). After transplantation, there was better graft function and less inflammatory response if a LF was used or a caspase-1 inhibitor was administered during EVLP. CONCLUSIONS: Our data demonstrated that circulating leukocytes derived from donor lungs, and not circulating proinflammatory cytokines substantially impaired the quality of lung grafts through caspase-1-induced pyroptotic cell death during EVLP. Removing these cells with a LF and/or inhibiting pyroptosis of the cells can be a new therapeutic approach leading to long-term success after lung transplantation.

Ex Vivo Lung Perfusion: A Key Tool for Translational Science in the Lungs.

Ex vivo lung perfusion (EVLP) promises to be a comprehensive platform for assessment, reconditioning, and preservation of donor lungs and has been dramatically changing the face of clinical lung transplantation. Besides its increasing role in lung transplantation, EVLP has also been recognized as a useful tool for translational research involving the lungs. Based on recent remarkable evidence and experience using EVLP in lung transplantation, there is growing interest in, and expectations for, the use of EVLP beyond the field of lung transplantation. By combining EVLP with advances in regenerative medicine, stem cell biology, and oncology, the evolving technology of EVLP has tremendous potential to advance pulmonary medicine and science. In this review, we revisit recent advances in EVLP technology and research and discuss the future translation of EVLP applications into life-changing medicine.