Optimal duration of ex vivo lung perfusion for heat stress-mediated therapeutic reconditioning of damaged rat donor lungs

Abstract

Objectives: Transient heat stress (HS) application during experimental ex vivo lung perfusion (EVLP) of warm ischaemic (WI) rat lungs produces a range of therapeutic benefits. Here, we explored whether different EVLP durations after HS application would influence its therapeutic effects.

Methods: In protocol 1, WI rat lungs were exposed to HS (41.5°C, 60-90 min EVLP), and EVLP was maintained for 3, 4.5 or 6 h (n = 5/group), followed by physiological measurements (compliance, oedema, oxygenation capacity). In protocol 2, WI rat lungs treated with (HS groups) or without HS (control groups) were maintained for 3 or 4.5 h EVLP (n = 5/group), followed by physiological evaluation and measurements (lung tissue) of heat shock proteins (HSP70, HSP27, HS90, GRP78), endogenous proteins (surfactant protein-D, CC16, platelet endothelial cell adhesion molecule-1), anti-apoptotic (Bcl2, Bcl-xL) and pro-apoptotic proteins (Bcl2-associated X protein, CCAAT/enhancer binding-protein homologous protein), antioxidant enzymes (heme-oxygenase-1, nicotinamide di-phospho-nucleotide dehydrogenase quinone-1) and nitrotyrosine (oxidative stress biomarker).

Results: In protocol 1, physiological variables were stable after 3 and 4.5 h but deteriorated after 6 h. In protocol 2, at 3 h EVLP, HS-treated lungs differed from controls by higher expression of HSP70 and heme-oxygenase-1, and lower CC16 expression. In contrast, at 4.5 h EVLP, HS-treated lungs displayed improved physiology, higher levels of all HSPs, preserved or increased expression of surfactant protein-D, CC-16 and platelet endothelial cell adhesion molecule-1, increased antioxidant and anti-apoptotic proteins, and reduced pro-apoptotic proteins and nitrotyrosine.

Conclusions: The protective effects of HS application during EVLP of WI-damaged rat lungs strictly depend on the duration of post-HS recovery. An EVLP duration of 4.5 h appears to optimize the therapeutic potential of HS, while maintaining lungs in a stable physiological state.

Keywords: Ex vivo lung perfusion; Animal model; Heat shock response; Heat stress reconditioning; Warm ischaemia.

Graphical abstract

Figure 1:

Experimental design. (A) Protocol 1: lungs exposed to 1 h warm ischaemia (WI) and 1 h cold ischaemia (CI) were perfused in an ex vivo lung perfusion (EVLP) system for 3, 4.5 or 6 h at 37°C, with the application of a transient heat stress (HS) at 41.5° from 1 to 1.5 h EVLP. (B) Protocol 2: lungs exposed to 1 h WI and 1 h CI were perfused in an EVLP system for 3 or 4.5 h, with (EVLP-HS) or without (EVLP-control) the application of a transient HS at 41.5° from 1 to 1.5 h EVLP.

Figure 2:

Pulmonary physiological variables following heat stress (HS) and ex vivo lung perfusion (EVLP) durations of 3 h, 4.5 h and 6 h. (A) Static pulmonary compliance (SPC), expressed as the ratio of SPC at the end of EVLP/1 h EVLP. (B) Maximal airway pressure (Pmax) expressed as the ratio of Pmax at the end of EVLP/1h. (C) Lung oedema development, assessed by the weight gain of the lungs at the end of EVLP. (D) Lung oxygenation capacity expressed in P/F ratio. N = 5 groups

Figure 3:

Lung physiological variables after 3 and 4.5 h ex vivo lung perfusion (EVLP). (A) Static pulmonary compliance (SPC), expressed as SPC ratio at the end of EVLP/1 h EVLP. (B) Maximal airway pressure (Pmax) expressed as the ratio of Pmax at the end of EVLP/1 h. (C) Lung oxygenation capacity expressed in P/F ratio. (D) Lung oedema development, assessed by the weight gain of the lungs at the end of EVLP. N = 5 groups.

Figure 4:

Lung expression of heat shock proteins at the end of 3 or 4.5 h ex vivo lung perfusion (EVLP). Lung tissue concentrations of (A) HSP70, (B) HSP27, (C) HSP90 and (D) HSPA5/GRP78. N = 5 groups.

Figure 5:

Expression of lung endogenous proteins at the end of 3 h or 4.5 h ex vivo lung perfusion (EVLP). (A) Surfactant protein D (SP-D), (B) Clara cell protein (CC16), (C) Platelet-endothelial cell adhesion molecule-1 (PECAM-1), all expressed in ng/mg or pg/mg lung tissue protein. (D) SP-D, (E) CC16, (F) PECAM-1, expressed as fold change in heat stress (HS) versus controls at each time-point. N = 5 groups.

Figure 6:

Pro- and anti-apoptotic proteins in lung tissue at 3 and 4.5 h ex vivo lung perfusion (EVLP). (A) B-cell leukemia/lymphoma-2-protein (Bcl-2), (B) B-cell lymphoma-extra large (Bcl-xL), (C) CCAAT/enhancer binding-protein (C/EBP) homologous protein (CHOP), expressed in ng/mg or pg/mg lung protein. Relative expression (fold changes in heat stress (HS) versus control lungs at 3 and 4.5 h EVLP) of (D) Bcl-2, (E) Bcl-xL, (F) CHOP. N = 5 groups.

Figure 7:

Antioxidant proteins and 3-nitrotyrosine in lungs at the end of ex vivo lung perfusion (EVLP) for 3 or 4.5 h. Lung tissue levels of (A) HO-1 (heme oxygenase-1), (B) NQO-1 [NAD(P)H quinone dehydrogenase 1], (C) 3-NT (3-nitrotyrosine). Relative expression [fold changes heat stress (HS) versus controls] of (D) HO-1, (E) NQO-1 and (F) 3-NT at 3 and 4.5 h EVLP. N = 5 groups.