Transient heat stress protects from severe endothelial damage and dysfunction during prolonged experimental ex-vivo lung perfusion

Abstract

Introduction: The pulmonary endothelium is the primary target of lung ischemia-reperfusion injury leading to primary graft dysfunction after lung transplantation. We hypothesized that treating damaged rat lungs by a transient heat stress during ex-vivo lung perfusion (EVLP) to elicit a pulmonary heat shock response could protect the endothelium from severe reperfusion injury.

Methods: Rat lungs damaged by 1h warm ischemia were reperfused on an EVLP platform for up to 6h at a constant temperature (T°) of 37°C (EVLP_37°C group), or following a transient heat stress (HS) at 41.5°C from 1 to 1.5h of EVLP (EVLP_HS group). A group of lungs exposed to 1h EVLP only (pre-heating conditions) was added as control (Baseline group). In a first protocol, we measured lung heat sock protein expression (HSP70, HSP27 and Hsc70) at selected time-points (n=5/group at each time). In a second protocol, we determined (n=5/group) lung weight gain (edema), pulmonary compliance, oxygenation capacity, pulmonary artery pressure (PAP) and vascular resistance (PVR), the expression of PECAM-1 (CD31) and phosphorylation status of Src-kinase and VE-cadherin in lung tissue, as well as the release in perfusate of cytokines (TNFα, IL-1β) and endothelial biomarkers (sPECAM, von Willebrand Factor -vWF-, sE-selectin and sICAM-1). Histological and immunofluorescent studies assessed perivascular edema and formation of 3-nitrotyrosine (a marker of peroxinitrite) in CD31 lung endothelium.

Results: HS induced an early (3h) and persisting expression of HSP70 and HSP27, without influencing Hsc70. Lungs from the EVLP_37°C group developed massive edema, low compliance and oxygenation, elevated PAP and PVR, substantial release of TNFα, IL-1β, s-PECAM, vWF, E-selectin and s-ICAM, as well as significant Src-kinase activation, VE-cadherin phosphorylation, endothelial 3-NT formation and reduced CD31 expression. In marked contrast, all these alterations were either abrogated or significantly attenuated by HS treatment.

Conclusion: The therapeutic application of a transient heat stress during EVLP of damaged rat lungs reduces endothelial permeability, attenuates pulmonary vasoconstriction, prevents src-kinase activation and VE-cadherin phosphorylation, while reducing endothelial peroxinitrite generation and the release of cytokines and endothelial biomarkers. Collectively, these data demonstrate that therapeutic heat stress may represent a promising strategy to protect the lung endothelium from severe reperfusion injury.

Keywords: animal model; ex-vivo lung perfusion; heat shock response; heat therapy; lung ischemia-reperfusion; lung transplantation; pulmonary endothelium.

Figure 1

Experimental protocols. In protocol 1, lungs exposed to 1h warm ischemia (WI) and 1h cold ischemia (CI) were maintained on EVLP for 1, 2, 3, 4.5 and 6h, either at constant T° of 37°C (EVLP_37°C group) or following transient heat stress at 41.5° for 30 min, starting at 1h EVLP (EVLP_HS group). At each time-point, lungs were obtained for HSP expression (n=5/group, except at 1h, n=5 in a single group). In protocol 2, lungs were maintained on EVLP for 1h (Baseline group, pre-heating conditions, n=5) or for 6h, either at constant T° of 37°C (EVLP_37°C group, n=5) or following transient heat stress as described above (EVLP_HS group, n=5). Measurements performed at the selected time-points are indicated. CI, Cold Ischemia; EVLP, Ex-Vivo Lung Perfusion; HS, Heat Stress; HSPs, Heat Shock Proteins; WI, Warm Ischemia.

Figure 2

Kinetics of Heat shock protein expression in rat lungs at different time points during EVLP. HSP70 (A), HSP27 (B) and HSC70 (C) in lung tissue homogenates, expressed in ng/mg lung protein. N=5/group at each time-point, except at 1h (one group only, n=5). * p < 0.05 vs EVLP_37°C; † p < 0.05 vs 1h EVLP.

Figure 3

Lung weight gain, oxygenation capacity and static compliance. (A) Lung weight gain between the start and the end of EVLP, measured as an index of lung edema, and (B) Oxygenation capacity (effluent perfusate P/FO₂ ratio) were determined after 1h EVLP (Baseline group, n=5) or after 6h EVLP in EVLP_37°C and EVLP_HS groups (n=5/group). (C) Static pulmonary compliance (SPC) was measured after 1 and 6h in EVLP_37°C and EVLP_HS (n=5/group). * p < 0.05 (inter-group difference); † p < 0.05 (6h vs 1h EVLP).

Figure 4

Lung morphology. (A) Representative macroscopic pictures of lungs from EVLP_37°C (upper images) and EVLP_HS (lower images) groups, after 1h and 6h EVLP. (B) Representative histological sections of lungs evaluated at 6h of EVLP. The black arrows indicate perivascular edema. The open arrow shows a bronchial structure. (C) Score of perivascular lung edema after 6h EVLP. N=5/group. * p < 0.05.

Figure 5

Pulmonary artery pressure and pulmonary vascular resistance during EVLP. (A) Pulmonary Artery Pressure and (B) Pulmonary Vascular Resistance at 1 and 6h EVLP in EVLP_37°C and EVLP_HS groups (n=5/group). * p < 0.05 (inter-group difference); † p < 0.05 (6h vs 1h EVLP).

Figure 6

Western immunoblot analysis of Src-kinase and VE-cadherin phosphorylation. Immunoblots (upper panel) and signal quantification (A–C) of total Src kinase, ^pTyr⁴¹⁹-Src, ^pTyr⁵²⁹-Src, total VE-cadherin, ^pTyr⁶⁸⁵-VE-cadherin, and β-actin (internal control) determined after 1h EVLP (Baseline group, n=5) or after 6h EVLP in EVLP_37°C and EVLP_HS groups (n=5/group). ∗p < 0.05.

Figure 7

Immunofluorescent detection of 3-nitrotyrosine and CD31. Representative sections of lungs from the EVLP_37°C (upper images) and EVLP_HS (lower images) groups after 6h EVLP. (A) CD31 immunostaining; (B) 3-NT immunostaining; (C) Merged images from (A, B) The regions encircled in the white squares in (C) are shown at higher magnification on the right. White arrows: alveolar structures; Arrowheads: extra-alveolar vessels; Large arrows: bronchioles. (D) Quantification of immunofluorescence staining of 3-NT formation in CD31 endothelium. (E) Histogram of the distribution of the data in each group. N=5/group. *p < 0.05.

Figure 8

Levels of inflammatory cytokines in the EVLP perfusate. (A) TNF-α and (B) IL-1β, measured in the EVLP perfusate at different time-points in the EVLP_37°C and EVLP_HS groups. N =5/group. * p < 0.05 vs EVLP_37°C; † p < 0.05 vs 1h EVLP.

Figure 9

Release of endothelial biomarkers during EVLP. (A) Von Willebrand Factor and (B) Soluble PECAM-1, measured at selected time-points in the perfusate from EVLP_37°C and EVLP_HS groups. (C) Lung tissue expression of PECAM-1 determined after 1h EVLP (baseline group) and after 6h EVLP in the EVLP_37°C and EVLP_HS groups. (D, E) Perfusate levels of Soluble E-selectin (D) and Soluble ICAM-1 (E) in EVLP_37°C and EVLP_HS groups. N=5/group. * p < 0.05 (intergroup differences); † p < 0.05 vs 1h EVLP.