5-Lipoxygenase deficiency prevents respiratory failure during ventilator-induced lung injury

Abstract

Rationale: Mechanical ventilation with high VT (HVT) progressively leads to lung injury and decreased efficiency of gas exchange. Hypoxic pulmonary vasoconstriction (HPV) directs blood flow to well-ventilated lung regions, preserving systemic oxygenation during pulmonary injury. Recent experimental studies have revealed an important role for leukotriene (LT) biosynthesis by 5-lipoxygenase (5LO) in the impairment of HPV by endotoxin.

Objectives: To investigate whether or not impairment of HPV contributes to the hypoxemia associated with HVT and to evaluate the role of LTs in ventilator-induced lung injury.

Methods: We studied wild-type and 5LO-deficient mice ventilated for up to 10 hours with low VT (LVT) or HVT.

Results: In wild-type mice, HVT, but not LVT, increased pulmonary vascular permeability and edema formation, impaired systemic oxygenation, and reduced survival. HPV, as reflected by the increase in left pulmonary vascular resistance induced by left mainstem bronchus occlusion, was markedly impaired in animals ventilated with HVT. HVT ventilation increased bronchoalveolar lavage levels of LTs and neutrophils. In 5LO-deficient mice, the HVT-induced increase of pulmonary vascular permeability and worsening of respiratory mechanics were markedly attenuated, systemic oxygenation was preserved, and survival increased. Moreover, in 5LO-deficient mice, HVT ventilation did not impair the ability of left mainstem bronchus occlusion to increase left pulmonary vascular resistance. Administration of MK886, a 5LO-activity inhibitor, or MK571, a selective cysteinyl-LT(1) receptor antagonist, largely prevented ventilator-induced lung injury.

Conclusions: These results indicate that LTs play a central role in the lung injury and impaired oxygenation induced by HVT ventilation.

Figure 1.

Survival during long-term mechanical ventilation in wild-type (WT) mice, during low VT (LVT; n = 12) or high VT (HVT; n = 15) ventilation, and in 5-lipoxygenase (5LO)–deficient mice (n = 8) and in MK886-pretreated WT mice (n = 12) during HVT ventilation (*p < 0.01 vs. other groups).

Figure 2.

The pressure–volume (PV) curve of the respiratory system in WT mice ventilated either (A) at LVT (n = 12) or (B) at HVT (n = 15), and in (C) 5LO-deficient mice (5LO^−/−; n = 8) and (D) MK886-pretreated WT mice (MK886; n = 12), during mechanical ventilation with HVT. Solid circles represent PV curve performed at baseline, open circles represent PV curve performed after 1 hour of ventilation, inverted solid triangles after 2 hours, inverted open triangles after 3 hours, solid squares after 4 hours, open squares after 5 hours, solid diamonds after 6 hours, open diamonds after 7 hours, solid hexagons after 8 hours, open triangles after 9 hours, and solid triangles represent PV curve performed at the end of the experiment. For clarity, only mean values are reported. PV curve values obtained at the end of the experiment represent mean values of the last PV curve performed in each animal (either after 10 hours of LVT ventilation or after a period between 7 and 10 hours of HVT mechanical ventilation). *p < 0.05 versus baseline; ^#p < 0.05 versus WT mice ventilated at LVT and 5LO-deficient and MK886-pretreated WT mice ventilated at HVT at the end of the experiment. IC = inspiratory capacity of the respiratory system, defined as the inflation volume needed to achieve 30 cm H₂O airway pressure.

Figure 3.

Extravascular lung water (EVLW) and alveolar–arterial oxygen tension difference (A-aDO₂; A) and total protein concentration in bronchoalveolar lavage fluid (BALF; B) in WT mice subjected to mechanical ventilation. After long-term mechanical ventilation, EVLW (n = 5) and A-aDO₂ (n = 15) were increased in mice ventilated with HVT compared with mice ventilated with LVT (n = 3 for EVLW, n = 12 for A-aDO₂) and mice studied at baseline (n = 3 for EVLW, n = 10 for A-aDO₂; *p < 0.01). Similarly, the total protein concentration in BALF was higher in mice after long-term HVT ventilation (n = 6) than in mice studied at baseline (n = 4), in mice after long-term LVT ventilation (n = 6), and in mice after ventilation with HVT (n = 5) for only 6 hours (*p < 0.001, all groups).

Figure 4.

Lung sections 2-μm thick from WT mice ventilated at LVT (A) and HVT (B), and from 5LO-deficient mice (C) and MK886-pretreated WT mice (D) both ventilated at HVT after long-term mechanical ventilation. Comparable areas from central lung regions are shown. Note the lacey network of open capillaries after (A) LVT ventilation (arrows) and the loss of these features after (B) HVT ventilation (arrows). The narrow alveolar–capillary membrane after HVT ventilation reflects capillary collapse and epithelial surface disruption. Note also the presence of neutrophils within the intravascular space (open arrow in B). The absence of these structural changes in 5LO-deficient mice (C) and WT mice pretreated with MK886 (D) demonstrates protection against injury induced by HVT ventilation. Bar = 25 μm; all other panels are at the same magnification. Alv = alveolar space.

Figure 5.

Effects of congenital deficiency of 5LO on the left mainstem bronchus occlusion (LMBO)–induced increase in left pulmonary vascular resistance (LPVR) in mice ventilated at LVT or HVT for 6 hours. In mice ventilated at LVT, LMBO markedly increased LPVR both in WT (n = 8) and 5LO-deficient mice (n = 4). In contrast, after HVT ventilation, LMBO did not increase LPVR in WT mice (n = 8), whereas it did increase LPVR in 5LO-deficient mice (n = 3; *p < 0.001 vs. WT mice). NS = not significant.

Figure 6.

Leukotriene (LT) C₄/D₄/E₄ (A) and LTB₄ (B) levels in BALF obtained from untreated and MK886-pretreated WT mice after long-term mechanical ventilation with LVT (n = 6, both groups) or HVT (n = 6, both groups) and from mice studied at baseline (n = 3, both groups). Both LTC₄/D₄/E₄ and LTB₄ levels were significantly increased in untreated WT mice after HVT ventilation (*p < 0.05 vs. LVT ventilation and baseline). In contrast, pretreatment with MK886, a 5LO-activating protein (FLAP) inhibitor, blocked the increase of LT production associated with HVT ventilation.

Figure 7.

Total protein concentration in BALF (A) and Pa_O2 (B) in untreated WT mice, 5LO-deficient mice, and WT mice treated with MK886 or MK571 after prolonged ventilation. Total protein concentration in BALF at the end of the experiment (A) was markedly higher in untreated WT mice ventilated at HVT (n = 6) than in WT mice ventilated at LVT (n = 6). Congenital deficiency of 5LO (n = 5) and treatment with MK886 (n = 6) or MK571 (n = 3) significantly reduced the increase in BALF total protein concentration caused by HVT ventilation (*p < 0.05, **p < 0.01 vs. LVT ventilation). Pa_O2 (B) markedly decreased in untreated WT mice after HVT ventilation (n = 10 at baseline, n = 5 at 6 hours, and n = 15 at the end of the experiment). In contrast, Pa_O2 remained unchanged during HVT ventilation in 5LO-deficient mice (n = 7 at baseline, n = 5 at 6 hours, and n = 8 at the end of the experiment), in MK886-pretreated WT mice (n = 6 at baseline, n = 5 at 6 hours, and n = 12 at the end of the experiment), and in MK571-treated WT mice (n = 5 at 6 hours, n = 6 at the end of the experiment).

Figure 8.

Neutrophil levels in BALF obtained from untreated WT mice, 5LO-deficient mice, and WT mice treated with MK886 studied at baseline (n = 4, each group) and after 6 hours of LVT or HVT ventilation (n = 5, each group), and from MK571-treated WT mice ventilated at HVT for 6 hours. HVT ventilation significantly increased neutrophil levels only in untreated WT and in MK571-treated WT mice (**p < 0.001 vs. baseline and LVT ventilation, *p < 0.05 vs. 5LO-deficient and MK886-pretreated WT mice ventilated at HVT). Congenital deficiency of 5LO and pretreatment with MK886 prevented the increased levels of neutrophils in BALF associated with HVT ventilation (p < 0.001 vs. untreated WT mice).