Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Jul 31;13(13):4376-4390.
doi: 10.7150/thno.87375. eCollection 2023.

Tackling regulated cell death yields enhanced protection in lung grafts

Qian Chen  1   2   3 , Xiangfeng Liu  2 , Zhiheng Liu  4 , Shujing Zhang  1 , Lin Chen  1 , Shiori Eguchi  1 , Azeem Alam  1 , Zhen Cahilog  1 , Qizhe Sun  1 , Lingzhi Wu  1 , Enqiang Chang  1 , Zhiping Wang  5 , Jianteng Gu  2 , Hailin Zhao  1 , Daqing Ma  1   3
Affiliations

Tackling regulated cell death yields enhanced protection in lung grafts

Qian Chen et al. Theranostics. .

Abstract

Background: Effective preservation strategies to ameliorate lung graft ischaemia injury are needed to rescue 'extended criteria' or 'marginal' lung grafts, and to improve recipient outcomes after transplantation. Methods: Lung grafts from male Lewis rats were extracted after 40 min of cardiocirculatory death, and healthy human lung tissues were collected from patients undergoing a lobectomy. Lung samples were then preserved in a 4°C preservation solution supplemented with 0.1 nM Dexmedetomidine (Dex, α2-adrenoceptor agonist) for 16 h. In vitro, human lung epithelial A549 cells were preserved in the 4°C preservation solution with 0.1 nM Dex for 24 h, then re-cultured in the cell culture medium at 37°C to mimic the clinical scenario of cold ischaemia and warm reperfusion. Lung tissues and cells were then analysed with various techniques including western blot, immunostaining and electron microscope, to determine injuries and the protection of Dex. Results: Prolonged warm ischaemia after cardiocirculatory death initiated Rip kinase-mediated necroptosis, which was exacerbated by cold storage insult and enhanced lung graft injury. Dex supplementation significantly reduced necroptosis through upregulating Nrf2 activation and reducing oxidative stress, thereby significantly improving lung graft morphology. Dex treatment also attenuated endoplasmic reticulum stress, stabilised lysosomes and promoted cell membrane resealing function, consequently reducing cell death and inflammatory activation after hypothermic hypoxia-reoxygenation in A549 cells. Conclusions: Inhibition of regulated cell death through Dex supplementation to the graft preservation solution improves allograft quality which may aid to expand the donor lung pool and enhance lung transplant outcomes per se.

Keywords: Dexmedetomidine.; Donation after cardiocirculatory death; Ischaemia reperfusion injury; Lung transplantation; Regulated cell death.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: Some data have been filed for a patent application.

Figures

Figure 1
Figure 1
Ischaemia induces lung graft injury and enhances inflammatory factors release in DCD lung grafts. Illustration of experimental procedure of ex vivo lung grafts: Lung graft from a living donation (LD) rat was excised directly after terminal anaesthesia. The lung graft from a donation after cardiac death (DCD) rat was extracted after 40 min of cardiac arrest. Both lung grafts were flushed and stored in the 4 °C UW solution for 0 or 16 h (CI0h or CI16h). (A) Histology (H&E staining) of lung grafts. Arrows indicate coagulative necrosis and cell deterioration, and areas marked by black circles indicate detachment of the dead cells. (B) Lung morphology was evaluated using a lung injury scoring system. (C) Lung tissue oxidative damage was detected with in situ OxyIHC oxidative stress detecting assay. (D) Lung tissue cell death was detected with in situ TUNEL assay. (E) The number of TUNEL+ cells. The concentration of (F) TNF-α, (G) IL-1b, and (H) HMGB-1 in the DCD lung graft tissue was assessed by ELISA. Lung grafts from rats that received PBS vehicle (Ve) or HMGB-1 (H) were extracted after 40 min of cardiac arrest and stored in the 4 °C UW solution for 16 h (CI16). (I) Histology (H&E staining) of the lung graft. (J) Lung injury score. Scale bar: 50 μm. Data are presented as scatter plot and expressed as median with interquartile range. n = 6. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 2
Figure 2
Dex supplementation suppresses necroptosis and inflammation in DCD lung grafts. Lung grafts were extracted from living donation (LD) and donation of cardiocirculatory death (DCD) donor rats and subsequently stored in a 4 °C preservation solution for 0 or 16 h (CI0h or CI16h). Labelling of (A) p-Rip3 (green) and p-MLKL (red) and fluorescent intensity of (B) p-Rip3 and (C) p-MLKL in lung tissues. Lung grafts were extracted after 40 min of DCD donors and stored in a 4 °C cold preservation solution supplemented with Dex (0.1 nM) or PBS (Ve) for 16 h (CI16h). Dual labelling of (D) p-Rip1 (green) and p-MLKL (red), p-Rip3 (green) and TLR-4 (red) in lung tissues. Fluorescent intensity of (E) p-Rip1, (F) p-Rip3, and (G) p-MLKL in lung tissues. (H) Histology (H&E staining) of lung grafts from DCD rats. (I) Lung morphology was evaluated using a lung injury scoring system. (J) Lung tissue cell death was detected with an in situ TUNEL assay. (K) The number of TUNEL+ cells. The concentration of (L) HMGB-1, (M) TNF-α, and (N) IL-1β in lung tissues were assessed by ELISA. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (blue). Scale bar: 50 μm. Data are presented as scatter plot and expressed as median with interquartile range. n = 6. *p < 0.05, **p < 0.01, ***p < 0.001. The smaller box view in the merged image with three immunofluorescent channels (right) was enlarged and presented with two-channel images individually on the left of each panel.
Figure 3
Figure 3
Dex activates the Nrf-2 pathway and suppresses oxidative stress in DCD lung grafts. Lung grafts were extracted after 40 min of cardiocirculatory death (DCD) and stored in a 4 °C cold preservation solution supplemented with Dex (0.1 nM) or PBS (Ve) for 16 h (CI16h). Normal lung grafts served as the naive control (NC). Dual labelling of (A) NQO-1 (green) and Nrf-2 (red), SOD-1 (green) and Nrf-2 (red) in lung tissue. Fluorescence intensity of (B) NQO-1, (C) Nrf-2, and (D) SOD-1 in lung tissue. (E) Lung tissue GSH level, (F) lung tissue GSSG level. (G) Lung tissue GSH to GSSG ratio. (H) Oxidative damage was evaluated by OxyIHC oxidative stress detecting assay. (I) The expression of 4-hydroxynonenal (green) and 3-nitrotyrosine (red) was assessed with immunofluorescent labelling. Fluorescent intensity of (J) 4-hydroxynonenal and (K) 3-nitrotyrosine. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (blue). Scale bar: 50 μm. Data are expressed as a scatter plot, median with interquartile range. n = 6. *p < 0.05, **p < 0.01, and ***p < 0.001. The smaller box view in the merged image with three immunofluorescent channels (right) was enlarged and presented with two-channel images individually on the left of each panel.
Figure 4
Figure 4
Nrf-2 suppression and α-adrenoceptor antagonism with atipamezole attenuate Dex-mediated rat lung graft protection. Lung grafts were extracted after 40 min of cardiocirculatory death and stored in a 4 °C UW cold storage solution saturated with Dex (0.1nM) or PBS (Ve) for 16 h (CI16h). Nrf-2 siRNA (NSi) or atipamezole (Atip) was given to the donor before graft extraction in comparison with scrambled siRNA (SSi) or PBS (Ve), respectively. Dual TLR-4 (red) and p-Rip-3 (green) were labelled in lung tissue after (A) siRNA or Atip treatment. Fluorescence intensity of (B) p-Rip3 and (C) TLR-4 in lung tissue. (D) The expression of 4-hydroxynonenal (green) and 3-nitrotyrosine (red) was assessed with immunofluorescent labelling. The expression of (E) 3-nitrotyrosine and (F) 4-hydroxynonenal was assessed. (G) Histology (H&E staining) of the lung graft and (H) injury score. (I) HMGB-1 in lung graft tissue was assessed by ELISA. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (blue). Scale bar: 50 μm. Data are expressed as scatter plot, median with interquartile range. n = 6. *p < 0.05, **p < 0.01 and ***p < 0.001. The smaller box view in the merged image with three immunofluorescent channels (right) was enlarged and presented with two-channel images individually on the left of each panel.
Figure 5
Figure 5
Dex supplementation protects human lung tissue during cold storage. Lung tissues from patients were cut into 3 pieces, 2 pieces were stored in a 4 °C UW solution saturated with Dex (0.1 nM) or PBS (Ve) for 16 h (CI16h) separately; another piece served as the naive control (NC). (A) Histology (H&E staining) of the lung tissue. (B) Lung morphology was evaluated using a lung injury scoring system. (C) Lung tissue cell death was detected by in situ TUNEL assay. (D) The number of TUNEL+ cells. Labelling of (E) p-MLKL (red) and fluorescent intensity of (F) p-MLKL in lung tissue. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (blue) or To-pro (red). Scale bar: 50 μm. Data are presented as scatter plots and expressed median with interquartile range. n = 10. *p < 0.05, **p < 0.01 and ****p < 0.0001. Transmission electron microscopy was performed on lung samples from patients No. 1, 3, and 6; (G) type II epithelial cells and (H) endothelial cells were observed. Asterisk: chromatin condensation; P: phagosome; LB: lamellar body; M: mitochondrial.
Figure 6
Figure 6
Hypothermic hypoxia-reoxygenation (HR) challenge induces necroptosis and plasma membrane rupture. Human lung epithelial A549 cells were preserved in a 0-4 °C UW solution for 24 h (H24h) to simulate hypothermia/hypoxia condition, then re-cultured in 37 °C fresh media to simulate reoxygenation. Labelling of p-MLKL (green), Annexin V (red), and CHMP4B (green) in A549 cells treated with (A) TLQ (TNF- α, 150 ng/mL; Q-VD-Oph, 40 µM; and LCL161, 10 µM) or (B) HR challenge. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (blue). Scale bar: 10 μm. (C) Western blot showing analysis (D) p-Rip3, (E) p-MLKL, and (F) NLRP3, GAPDH as a loading control. n = 5-7. A549 cells were preserved in 0-4 °C UW solution for 24 h, then probed with 2 μM calcium indicator Fura-2 for 30 min and cultured in 37 °C media. The fluorescence intensity (G) of Fura-2 of 90 cells was calculated in each group. n = 5. Data are presented as scatter plot and median with interquartile range, or mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.
Figure 7
Figure 7
Dex supplementation reduces oxidative stress and PANoptosis after hypothermic hypoxia-reoxygenation (HR). A549 cells were stored in a 4 °C UW solution saturated with or without Dex (0.1 nM) or /and atipamezole (Atip) for 24 h and were then cultured in 37 °C media for another 6 h. (A) Western Blot analysis of (B) GSDMD, (C) Caspase-1 (Casp-1), (D) Caspase-5 (Casp-5), (E) Caspase-3 (Casp-3), (F) Caspase-8 (Casp-8), (G) p-MLKL, (H) p-Rip, (I-J) the expression of NLRP3 in A549 cells experienced HR. (K) Membrane lipid was stained by DilC18 dye. (L) Dual labelling of LAMP1 (yellow) and Grp78 (green), LAMP1 (yellow) and CHOP (green) in A549 cells. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (blue). Fluorescence intensity of (M) Grp78 and (N) CHOP in A549 cells. Data are presented as scatter plot and expressed as median with interquartile range. Scale bar: 10 μm. n = 4-8. *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001.
Figure 8
Figure 8
Molecular mechanisms of Dexmedetomidine-mediated protection to lung grafts. In the DCD setting, the inevitable warm ischemia during cardiocirculatory arrest and organ extraction leads to endoplasmic reticulum stress and oxidative stress that initiates necroptosis and apoptosis. The following cold storage means a lack of ATP production by oxidative phosphorylation. However, ATP demanding process continues because cold tolerance is finite; metabolism may slow but does not entirely stop at low temperatures. These will cause energy depletion, and hence ions to pump failure exacerbate cell death. Furthermore, the damage to cells upon reperfusion releases damage-associated molecular patterns (DAMPs) and pro-inflammatory cytokines, which initiate PANoptosis. Pore-forming proteins, such as GSDMD and MLKL in PANoptotic cells, lead to plasma membrane rupture and allow cytoplasmic outflow, which activates the innate immune response and enhances inflammatory damage to the tissue, then contributes to organ dysfunction and rejection. Dexmedetomidine (Dex) treatment in the ex vivo cold-storing stage inhibited oxidative stress and the following necroptosis. In addition, Dex can stabilise lysosomes during cold storage and promote membrane repair after reperfusion, maintain cell membrane integrity, and suppresses PANoptosis core proteins. These protective effects preserve lung epithelial cells and DCD lung graft function after engraftment per se.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Laubach VE, Sharma AK. Mechanisms of lung ischemia-reperfusion injury. Curr Opin Organ Transplant. 2016;21:246–52. - PMC - PubMed
    1. Thabut G, Mal H. Outcomes after lung transplantation. J Thorac Dis. 2017;9:2684–91. - PMC - PubMed
    1. Valapour M, Lehr CJ, Skeans MA, Smith JM, Uccellini K, Goff R. et al. OPTN/SRTR 2018 Annual Data Report: Lung. Am J Transplant. 2020;20(Suppl s1):427–508. - PubMed
    1. Weill D. Lung transplantation: indications and contraindications. J Thorac Dis. 2018;10:4574–87. - PMC - PubMed
    1. Reeb J, Keshavjee S, Cypel M. Expanding the lung donor pool: advancements and emerging pathways. Curr Opin Organ Transplant. 2015;20:498–505. - PubMed

Publication types