Activation of p21 limits acute lung injury and induces early senescence after acid aspiration and mechanical ventilation

Abstract

The p53/p21 pathway is activated in response to cell stress. However, its role in acute lung injury has not been elucidated. Acute lung injury is associated with disruption of the alveolo-capillary barrier leading to acute respiratory distress syndrome (ARDS). Mechanical ventilation may be necessary to support gas exchange in patients with ARDS, however, high positive airway pressures can cause regional overdistension of alveolar units and aggravate lung injury. Here, we report that acute lung injury and alveolar overstretching activate the p53/p21 pathway to maintain homeostasis and avoid massive cell apoptosis. A systematic pooling of transcriptomic data from animal models of lung injury demonstrates the enrichment of specific p53- and p21-dependent gene signatures and a validated senescence profile. In a clinically relevant, murine model of acid aspiration and mechanical ventilation, we observed changes in the nuclear envelope and the underlying chromatin, DNA damage and activation of the Tp53/p21 pathway. Absence of Cdkn1a decreased the senescent response, but worsened lung injury due to increased cell apoptosis. Conversely, treatment with lopinavir and/or ritonavir led to Cdkn1a overexpression and ameliorated cell apoptosis and lung injury. The activation of these mechanisms was associated with early markers of senescence, including expression of senescence-related genes and increases in senescence-associated heterochromatin foci in alveolar cells. Autopsy samples from lungs of patients with ARDS revealed increased senescence-associated heterochromatin foci. Collectively, these results suggest that acute lung injury activates p53/p21 as an antiapoptotic mechanism to ameliorate damage, but with the side effect of induction of senescence.

Fig 1

Expression of gene signatures. A, Overview of the analysis. Eleven datasets (128 samples) reporting gene expression in animal models of lung injury were pooled and analyzed to calculate different Meta-scores summarizing the expression of genes included in specific signatures. B, Meta-score of a p53-dependent signature for each experimental group (second hit refers to any model of lung injury other than mechanical ventilation). C, Meta-score of a transcriptomic signature including genes downregulated by p21. D, Meta-score of a senescence-specific signature. Gray lines mark significant differences among groups (P < 0.05 in Tukey's post hoc tests).

Fig 2

Characterization of lung injury. A, Acid instillation and mechanical ventilation caused lung damage assessed using a histological score (scale bar:100 μ). B, Myeloperoxidase-positive cell counts in histological sections, showing an increase of neutrophils in the injured lung. C, Expression of Il6 in lung tissue. D, Quantification of Ki-67 positive cells in histological sections, as a marker of proliferation. E, TUNEL-positive cells in histological sections. F, Abundance of cleaved Caspase-9 in lung homogenates. G–H, Changes in Lamin-A/Lamin-B1 ratio in nuclei from lung tissue (G) and representative immunohistochemical sections (H, scale bar: 25 μ). (I–J) Abundance of γH2AX (I) and HP1α (J), markers of DNA damage and heterochromatin respectively, in nuclei from lung tissue. K, Representative western blots of the previous quantifications. L–O, Changes in expression of the canonical senescence inducers Cdkn2a (p16, L), Rb (M), Tp53 (N) and Cdkn1a (p21, O). N = 4–6 animals per group. Gray lines mark significant differences among groups (P < 0.05 in Tukey's post hoc tests).

Fig 3

Lung injury in wildtype and Cdkn1a^−/− animals. A, Histological score of lung damage in both genotypes (scale bar: 100 μ). B, Percentage of apoptotic (TUNEL+) cells (scale bar: 50 μ). C, Abundance of cleaved caspase-9 in lung homogenates from both genotypes. D–E, Expression of Il6 (D) and Tp53 (E) in wildtype and mutant mice. F–G, Abundance of γH2AX (F) and HP1α (G), with representative western blots, in lung homogenates. N = 4–6 animals per group. Gray lines mark significant differences among groups (P < 0.05 in T tests).

Fig 4

Effects of Lopinavir/Ritonavir on lung injury. A, Lamin-A abundance and staining in vehicle- and lopinavir/ritonavir treated animals (scale bar: 25 μ). B, Histological score of lung damage (scale bar: 100 μ). C, Apoptotic (TUNEL+) cell counts in both groups (scale bar: 50μ). D-F, Abundance of Caspase-9 in tissue homogenates (D), γH2AX (E) and HP1α (F), with representative western blots (G) in lung homogenates. H–J, Expression of Il6 (H), Tp53 (I) and Cdkn1a (p21, J). N = 7–10 animals per group. Gray lines mark significant differences among groups (P < 0.05 in T tests).

Fig 5

Identification of early senescence markers in experimental models and patients. A, Counts of Senescence-associated heterochromatin foci (SAHF) in the experimental model of lung injury of acid instillation and mechanical ventilation (scale bar: 50μ). B–D, Expression of Plk3, Gdnf, and Meis1 in lung tissue. These senescence-associated genes were identified in the genomic analysis as those with the largest differences between control and injured samples. E–F, SAHF counts (E) and Plk3 expression (F) in wildtype and Cdkn1a^−/− mice after lung injury. G–H, SAHF counts (G, scale bar: 25μ) and Plk3 expression (H) in vehicle and lopinavir/ritonavir (LPV)-treated mice after lung. I, Appearance of SAHF in autopsy samples from critically ill patients who died in the Intensive Care Unit with or without mechanical ventilation and acute respiratory distress syndrome (ARDS) (scale bar: 50μ). N = 4–7 animals per group, with 3 slides per animal as technical replicates in SAHF counts. Gray lines mark significant differences among groups (P < 0.05 in Tukey's post hoc or in T tests).

Fig 6

The role of p21 pathway on apoptosis and senescence after acute lung injury. A, In control mice, lung injury and mechanical stretch cause DNA damage and changes in the nuclear envelope, activating the cell senescence program. The amount of apoptotic cells depends on the equilibrium between the activation of proapoptotic responses triggered by injury itself and the antiapoptotic effects of the senescence inducer Cdkn1a (p21). B, In mice lacking Cdkn1a, absence of this antiapoptotic factor leads to an increase in apotosis and a more severe lung injury. C, Treatment with Lopinavir/ritonavir blocks the Lamin-A mediated chromatin remodeling, triggering a senescence-like response that increases p21 expression, thus decreasing apoptosis and lung damage.