Despite best efforts, there remains much to improve in both the short-term and long-term outcomes of lung transplantation (LTx), particularly when compared with transplantation of other organs such as the heart. Although there may be a multitude of reasons for this, the effects of ischemia reperfusion injury (IRI), to which the lung is particularly sensitive, is a particular concern. Mediated by oxidative stress leading to a damaging inflammatory response, in the setting of LTx, IRI is associated with both primary graft dysfunction, the most common cause of death in the immediate posttransplant period, and poorer long-term outcomes.1
Ex vivo lung perfusion (EVLP) allows more comprehensive assessment of procured organs and results in a greater acceptance rate. Overall, wait times for LTx may be reduced, meaning fewer individuals die before they can be offered donor organs.2 IRI can occur on initiation of EVLP, and EVLP can be used as a research tool as well as for clinical purposes.
Little progress has been made in preventing or treating IRI, although some ideas, such as interleukin-10 gene therapy, have shown some promise in preclinical studies.3 Enhancing the knowledge of IRI may identify biomarkers and uncover new possibilities for therapeutic modulation.
Now, in Transplantation, Elgharably et al4 expand this field to include study of levels of microribonucleic acids (miRNAs) using EVLP to simulate IRI in human lungs.
MiRNAs are small, noncoding ribonucleotide strands that posttranscriptionally regulate gene expression (Figure 1). Work in the field of miRNAs has opened up a whole new field for the mechanistic understanding of a wide range of physiological and pathological settings and of drug effects.5
In presenting their elegant study, Elgharably et al advance the field in 2 main ways.
First, they demonstrate the feasibility of EVLP as a method to study miRNA expression of perfused human lungs. The implication is that this technique might help us to study a wide range of physiological and pathological processes mediated and marked by miRNAs in the lung allograft. This might include, as in this case, those relevant to IRI, but potentially many other areas too. They demonstrate measurements in perfusate, which may be extrapolated to circulating levels in vivo.
Second, they identified that levels of a number of miRNAs appeared different between controls and in ischemic and reperfusion settings. Two in particular stood up to validation with quantitative polymerase chain reaction and in situ hybridization, namely, miR-17 and miR-548b. These 2 appear significantly upregulated during both cold ischemia and reperfusion when compared with controls. Although always difficult to discern between cause and effect, these miRNAs do have a proven role in regulating factors relating to lung injury, for example, both miR-17 and miR-548b are predicted to regulate expression of important proinflammatory cytokines such as interleukin-6 and tumor necrosis factor α, proapoptotic factors such as caspase 9, and damage receptors such as toll-like receptor 4, all known to have a role in primary graft dysfunction.4 Though not yet definitively established, causality therefore has the backing of biological plausibility.
Although this is the first full article to describe miRNA profiles in human lung IRI, this has been characterized in other species and for other organs, with some contrasting findings to the present study. For example, in murine lung IRI, miR-223 was identified as a key regulator, while in murine cardiac IRI, miRNAs not including miR-17 or miR-548b appeared significant.6,7 Both interspecies variation and the known differences in cytokine profiles during IRI of different organs may account for some of these differences.
What might this mean for the battle against LTx-related IRI? If allowed to speculate, we might imagine 2 main possibilities.
First, use of miRNAs as clinically useful biomarkers is an emerging field. Developing new biomarker tests is difficult because of the need to be sensitive, specific, and reliable. If considering a one-off measurement as a clinical test, there needs to be good separation of the values between the positive and negative groups, rather than simply a significant difference. However, techniques such as measuring proportional changes over time, rather than one-off values, or assimilating measurements of a number of different markers, might make interpretation easier. If an miRNA marker of IRI offers more specificity than, for example, levels of cytokines that are likely to be elevated for a range of reasons in the posttransplant setting, this would also offer advantages.
Obviously, there are many steps and potential pitfalls before this could be clinically applicable, including correlation with clinical outcomes, but there is no doubt that a well-validated new biomarker for IRI would have obvious value.
While assessing the degree of IRI during EVLP may greatly aid graft assessment, detecting it after LTx is less useful if there are no specific treatments to call upon.
Evidence of the upregulation of miRNAs known to have roles in lung damage also presents potential therapeutic targets. Specific miRNAs present particularly powerful foci given they are typically involved in regulating a multitude of factors in a biological network (Figure 1). While in its relative infancy, the development of antagonists to miRNAs, known as antagomirs, is becoming established, having first been described in 2005. Antagomirs take the form of antisense oligonucleotides engineered to resist the physiological rapid degradation of RNA. They have been developed and investigated in a range of settings including lung diseases such as pulmonary fibrosis, bronchial hypersensitivity, and lung injury, although none have yet reached clinical studies.8 Notably, however, antagomir-based therapy has begun to reach patient-level trials in other disease areas. Miraversen is an antagomir of miR-122, which has been implicated in the propagation of hepatitis C virus. Phase 2 studies have demonstrated a reduction in detectable levels of hepatitis C virus, and the drug appeared safe and well tolerated, proving the feasibility and potential efficacy of this strategy.9 It remains to be seen whether this and other antagomirs are eventually introduced into routine clinical practice in any area, let alone in the setting of LTx-associated IRI. The unsurprising fact that multiple miRNA appear to be involved in IRI reduces the chance of a single “magic bullet,” but on the other hand, the finding that 2 appear most prominent is promising given there is already evidence from animal studies that dual targeting of 2 specific miRNA may reduce IRI in other organs, for example, in renal grafts.10
Of course, there are great limitations to the extrapolations we can make from a study of this kind. However, the authors have done a good job of acknowledging these drawbacks, and of course, while clearly more work is needed, it does represent a new and potentially promising area for study.
Although the transplant community continue to struggle with the specter of IRI and its consequences, at least avenues for the exploration of new approaches continue to appear.
1. Laubach VE, Sharma AK. Mechanisms of lung ischemia-reperfusion injury. Curr Opin Organ Transplant. 2016; 21:246–252
2. Okamoto T, Wheeler D, Farver CF, et al. Transplant suitability of rejected human donor lungs with prolonged cold ischemia time in low-flow acellular and high-flow cellular ex vivo lung perfusion systems. Transplantation. 2019; 103:1799–1808
3. Kozower BD, Kanaan SA, Tagawa T, et al. Intramuscular gene transfer of interleukin-10 reduces neutrophil recruitment and ameliorates lung graft ischemia-reperfusion injury. Am J Transplant. 2002; 2:837–842
4. Elgharably H, Okamoto O, Ayyat KS, et al. Human lungs airway epithelium up-regulate MicroRNA-17 & MicroRNA-548b in response to cold ischemia & ex-vivo reperfusion Transplantation. 2020; 104:1842–1852
5. Anglicheau D, Muthukumar T, Suthanthiran M. MicroRNAs: small RNAs with big effects. Transplantation. 2010; 90:105–112
6. Zhou L, He M, Mo Z, et al. A genome wide association study identifies common variants associated with lipid levels in the Chinese population. PLoS One. 2013; 8:e82420
7. Ye C, Qi W, Dai S, et al. microRNA-223 promotes autophagy to aggravate lung ischemia-reperfusion injury by inhibiting the expression of transcription factor HIF2α Am J Physiol Lung Cell Mol Physiol. [Epub ahead of print. April 8, 2020] doi: 10.1152/ajplung.00009.2020
8. Murdaca G, Tonacci A, Negrini S, et al. Effects of antagomiRs on different lung diseases in human, cellular, and animal models Int J Mol Sci. 2019; 20:3938. doi: 10.3390/ijms20163938
9. Janssen HL, Reesink HW, Lawitz EJ, et al. Treatment of HCV infection by targeting microRNA. N Engl J Med. 2013; 368:1685–1694
10. Tingle SJ, Sewpaul A, Bates L, et al. Dual microRNA blockade increases expression of antioxidant protective proteins: implications for ischaemia reperfusion injury Transplantation. 2020; 104:1853–1861