Research ArticleLUNG VENTILATION

Preventing loss of mechanosensation by the nuclear membranes of alveolar cells reduces lung injury in mice during mechanical ventilation

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Science Translational Medicine  29 Aug 2018:
Vol. 10, Issue 456, eaam7598
DOI: 10.1126/scitranslmed.aam7598
  • Fig. 1 Scaling of nuclear lamins with driving pressures in alveolar epithelium.

    (A) Representative Western blot showing Lamin-A, Lamin-B, and Lamin-C abundance during spontaneous breathing (baseline) or during mechanical ventilation with different driving pressures (15 and 20 cmH2O) [means ± SEM, P value obtained using one-way analysis of variance (ANOVA); brackets indicate significant post hoc comparisons using a Tukey’s honest significant difference (HSD) test; n = 5 per condition]. (B and C) Bar graph showing Lamin-A/Lamin-B ratio (B; n = 5 per condition) and Lmna gene expression (C; n = 8 per condition) at baseline and during mechanical ventilation. Data are shown as means ± SEM, P values obtained using a t test. (D) Representative immunofluorescence of lungs from spontaneous breathing (Baseline) and mechanically ventilated mice (with a driving pressure of 15 cmH2O). DAPI, 4′,6-diamidino-2-phenylindole. (E) Bar graphs showing LAMIN-A (top) and LAMIN-B (bottom) expression in airway epithelium, endothelium, and alveoli in lung samples from autopsies of patients who died during spontaneous breathing (n = 5) or mechanical ventilation alone or in combination with ARDS (n = 5 and n = 7, respectively). Data are shown as means ± SEM; P values indicate comparison between ventilated and nonventilated patients using two-way ANOVA including lung injury and ventilation as factors, followed by Tukey’s HSD post hoc test when significant (brackets). (F) Representative histology of alveoli showing LAMIN-A and LAMIN-B expression in patients described in (E). (G) Schematic representation of nuclear compliance measurements using micropipette aspiration. (H) Changes in compliance of nuclei (means ± SEM; n = 3 per group) from BEAS-2b cells at different times after stretch and recovery (P values obtained in the repeated-measurements ANOVA; asterisks represent a P value lower than 0.05 in Tukey’s HSD post hoc tests when compared to baseline values).

  • Fig. 2 Lung injury in Zmpste24-deficient mice.

    (A and B) Bar graphs showing lung damage after mechanical ventilation in wild-type and Zmpste24−/− mice, measured by a histological scale (A) or the albumin content of bronchoalveolar lavage fluid (BALF; B). P values obtained using two-way ANOVA including genotype and ventilation as factors; brackets indicate significant post hoc comparisons using a Tukey’s HSD test; n = 8 per condition and genotype. (C) Representative histological lung sections. (D and E) Values of arterial PO2 (D) and lung compliance (E) after ventilation with a driving pressure of 15 cmH2O. P value obtained using a t test; n = 4 per genotype and condition. (F) Representative Western blot showing the presence of Prelamin-A in mutant mice compared to the mature form observed in wild-type animals. (G) Quantification of Prelamin-A/Lamin-B ratio in Zmpste24−/− mice in baseline conditions or after ventilation with 15 or 20 cmH2O driving pressure (n = 5 per group; P value obtained using one-way ANOVA). (H) Graphs showing nuclear compliance measured by micropipette aspiration of nuclei from lungs of wild-type and Zmpste24−/− mice in baseline conditions and after mechanical ventilation (n = 6 per condition and genotype; P values obtained using one-way ANOVA; brackets show P values of Tukey’s HSD post hoc comparisons). (I) Representative images of electron microscopy showing chromatin rearrangement in response to mechanical ventilation in nuclei from wild-type and mutant animals. (J and K) Expression of the mechanosensitive genes Egr1 (H) and Ier3 (I) [P values obtained using two-way ANOVA including genotype and ventilation as factors, followed by Tukey’s HSD post hoc test when significant (brackets); n = 8 per condition and genotype]. (L) Effects of silencing ZMPSTE24 or LMNA expression in human bronchial cells subjected to stretch compared to pretreatment with a sham small interfering RNA (siRNA) [P values obtained using one-way ANOVA, followed by Tukey’s HSD post hoc test (brackets), are depicted in the graph; n=3 per group]. All data are shown as means ± SEM.

  • Fig. 3 Differential gene expression in lungs from Zmpste24−/− mice subjected to mechanical ventilation.

    (A) Venn’s diagram showing the number of genes with significantly different expression after mechanical ventilation in wild type and Zmpste24−/− mice. (B) Heat map of the 200 genes with the top differential expression. (C) Graph showing the number of genes and the significance level of biological processes corresponding to the differentially expressed genes, according to the Ingenuity Pathway Analysis. NFκB, nuclear factor κB. (D) Bar graph showing the percentage of apoptotic cells in response to ventilation in wild-type and Zmpste24−/− mice. P values obtained using two-way ANOVA including genotype and ventilation as factors, followed by Tukey’s HSD post hoc test when significant (brackets), and representative images of TUNEL staining. All data are shown as means ± SEM unless otherwise specified.

  • Fig. 4 Effects of lopinavir/ritonavir in VILI and nuclear compliance.

    (A) Representative Western blot of nuclear lamins in vehicle- and lopinavir/ritonavir-treated animals subjected to mechanical ventilation. (B) Bar graph showing changes in Lamin-A/Lamin-B ratio with treatment. P values obtained using two-way ANOVA including treatment and ventilation as factors, followed by Tukey’s HSD post hoc test when significant (brackets); n = 4 per condition and treatment. (C) Immunofluorescence images illustrating the organization of nuclear Lamin-A after an intraperitoneal injection of lopinavir/ritonavir or its vehicle. (D) Nuclear compliance after mechanical ventilation in vehicle- and lopinavir/ritonavir-treated animals (P = 0.003 for the effect of treatment obtained using a repeated-measurements ANOVA, including ventilation, treatment, and time as factors, followed by Tukey’s HSD test, depicted in the graph; n = 4 per condition and treatment). (E) Effects of treatment on Egr1 gene expression. P values obtained using two-way ANOVA including treatment and ventilation as factors, followed by Tukey’s HSD post hoc test when significant (brackets); n = 4 per condition and treatment. (F to I) Quantification of lung damage after ventilation in animals treated with vehicle or lopinavir/ritonavir using a histological score (F; n = 8 per condition and treatment; panels show representative sections stained with hematoxylin and eosin), albumin concentration in bronchoalveolar lavage fluid (G; n = 5 per group), PaO2 (H; n = 4 per group), and lung compliance at the end of ventilation (I; n = 4 per group). P values obtained using t tests. (J) Apoptotic cell count after ventilation in vehicle- and lopinavir/ritonavir-treated mice (n = 3 per condition and treatment) and representative TUNEL staining in each experimental group. P values obtained using two-way ANOVA including treatment and ventilation as factors, followed by Tukey’s HSD post hoc test when significant (brackets). All data are shown as means ± SEM.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/456/eaam7598/DC1

    Fig. S1. Changes in nuclear lamins with ventilation.

    Fig. S2. Lamin-A and Lamin-B in airway epithelium and lung endothelium in patients.

    Fig. S3. Changes in lamin content and nuclear compliance in epithelial cells and fibroblasts.

    Fig. S4. Stretch-induced lung injury in Lamin-A–deficient mice (LmnaLCS/LCS).

    Fig. S5. Differences in the inflammatory response to ventilation between genotypes.

    Fig. S6. Prelamin-A and Lamin-B abundance in Zmpste24−/− mice subjected to mechanical ventilation.

    Fig. S7. Prelamin-A and Lamin-A staining after ventilation.

    Fig. S8. Endotoxin-induced lung injury in wild-type and Zmpste24−/− mice.

    Fig. S9. Principal components analysis of the microarray data.

    Fig. S10. Differential mechanosensitive gene expression in wild-type and Zmpste24−/− mice subjected to mechanical ventilation.

    Fig. S11. Differential expression of genes involved in apoptosis.

    Fig. S12. Apoptosis after LPS injection in wild-type and Zmpste24−/− mice.

    Fig. S13. Effects of HIV protease inhibitors in LmnaLCS/LCS mice.

    Fig. S14. Effects of HIV protease inhibitors in Zmpste24−/− mice.

    Fig. S15. Effects of lopinavir/ritonavir on the repair phase after VILI.

    Fig. S16. Genetic and pharmacological blockade of the mechanical response to mechanical ventilation.

    Fig. S17. Respiratory mechanics at baseline.

    Table S1. Patients’ characteristics.

    Table S2. Statistical power of nonsignificant comparisons.

    Table S3. Raw data.

  • The PDF file includes:

    • Fig. S1. Changes in nuclear lamins with ventilation.
    • Fig. S2. Lamin-A and Lamin-B in airway epithelium and lung endothelium in patients.
    • Fig. S3. Changes in lamin content and nuclear compliance in epithelial cells and fibroblasts.
    • Fig. S4. Stretch-induced lung injury in Lamin-A–deficient mice (LmnaLCS/LCS).
    • Fig. S5. Differences in the inflammatory response to ventilation between genotypes.
    • Fig. S6. Prelamin-A and Lamin-B abundance in Zmpste24−/− mice subjected to mechanical ventilation.
    • Fig. S7. Prelamin-A and Lamin-A staining after ventilation.
    • Fig. S8. Endotoxin-induced lung injury in wild-type and Zmpste24−/− mice.
    • Fig. S9. Principal components analysis of the microarray data.
    • Fig. S10. Differential mechanosensitive gene expression in wild-type and Zmpste24−/− mice subjected to mechanical ventilation.
    • Fig. S11. Differential expression of genes involved in apoptosis.
    • Fig. S12. Apoptosis after LPS injection in wild-type and Zmpste24−/− mice.
    • Fig. S13. Effects of HIV protease inhibitors in LmnaLCS/LCS mice.
    • Fig. S14. Effects of HIV protease inhibitors in Zmpste24−/− mice.
    • Fig. S15. Effects of lopinavir/ritonavir on the repair phase after VILI.
    • Fig. S16. Genetic and pharmacological blockade of the mechanical response to mechanical ventilation.
    • Fig. S17. Respiratory mechanics at baseline.
    • Table S1. Patients’ characteristics.
    • Table S2. Statistical power of nonsignificant comparisons.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S3 (Microsoft Excel format). Raw data.

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