Increased phospholipase A2 and lyso-phosphatidylcholine levels are associated with surfactant dysfunction in lung contusion injury in mice

Abstract

Objective: Surfactant dysfunction is an important pathologic disturbance in various forms of acute inflammatory lung injury. Previously we reported the presence of marked alterations in the composition and activity of pulmonary surfactant in bilateral lung contusions (LC) injury induced by blunt trauma in rats. This is extended here to a mouse model of unilateral LC with a focus on compositional and functional changes in surfactant associated with permeability injury and increases in activity of secretory phospholipase A2.

Results: Surfactant-associated gene expression was not altered in mice with unilateral LC injury on the basis of Affymetrix analysis. LC mice had significant permeability injury with increased albumin and total protein in bronchoalveolar lavage at 5, 24, 48, and 72 hours after insult compared with uninjured controls. The percent content of large surfactant aggregates was depleted at all postinjury times, and pulmonary pressure-volume (P-V) mechanics and compliance were abnormal during this period. Surfactant dysfunction was evaluated in 24 hours, when permeability injury and P-V changes were most prominent. At this time, activity levels of secretory phospholipase A2 were increased in bronchoalveolar lavage, and chromatographic analysis showed that large surfactant aggregates had decreased levels of phosphatidylcholine and increased levels of lyso-phosphatidylcholine. These changes were accompanied by severe detriments in large aggregate surface activity by pulsating bubble surfactometry. Large aggregates from LC mice at 24 hours had minimum surface tensions of only 12.6 ± 1.1 mN/m after prolonged bubble pulsation (20 min) compared with 0.7 ± 0.03 mN/m for uninjured controls.

Conclusion: These results document important detriments in the composition and activity of pulmonary surfactant in LC injury in mice and suggest that active synthetic phospholipase-resistant exogenous surfactants may have utility in treating surfactant dysfunction in this clinically important condition.

FIGURE 1. Potential mechanism of Lung Contusion (LC) induced alteration on surfactant composition and activity

After lung contusion, increased activation of Phospholipase A₂ (PLA₂) will degrade surfactant phospholipids such as Phosphatidylcholine into Lyso-phosphatidylcholine, modifying the qualities of surfactant and directly increasing inflammation. Additionally the loss of large aggregates due to interactions with plasma proteins coming from permeability injury and due to increased activity of PLA₂ will diminish surfactant tension-reducing properties having downstream effects in lung mechanics (i.e reducing compliance).

FIGURE 2. Large surfactant aggregate content in bronchoalveolar lavage (BAL) during evolving lung contusion

Large aggregates were centrifuged at 12,000 × g from cell-free BAL from uninjured control mice and LC mice at 5, 24, 48, and 72 h post-contusion and subsequently quantitated by phosphate assay as a percentage of total lavage phospholipid. Surfactant aggregates were found to be depleted most prominently early in LC injury (5 and 24 h), but remained depleted below control levels throughout the 72 h time period studied. ⁺ p < 0.05 compared to uninjured control; * p < 0.001 or less compared to uninjured control; # p < 0.01 or less compared to 24 h injury. Data are Mean ± S.E.M. with N= 5–9 except for injured mice at 5 h (N=2).

FIGURE 3. Pressure-volume (P-V) curves in evolving lung contusion injury in mice

Pressure-volume curves were generated with the PVrV force oscillation maneuver using a SCIREQ Flexivent® mice ventilator. Compliance was diminished in all post-injury time points compared to uninjured controls, with the greatest decrease in P-V compliance seen at 24 h post-injury. Curves were generated with over 4000 data points and provide a visual for the overall observation of these curves, similar to observations in the currently used mechanical ventilators. For detailed statistical analysis and interpretations from the PV curves, please refer to Figure 3. Tick marks represent major integral of pressures during inflation and deflation.

FIGURE 4. Maximal lung volumes, hysteresis and respiratory mechanics in lung contusion

A. Volume obtained using a standard inspiratory pressure of 30 cm H₂O (TLC-30). TLC-30 was significantly diminished at 24, 48 and 72 h post-injury compared to uninjured control; B. Maximal volume for a single ramped breath (Vend) indicative of Quasi-static compliance, showing significantly diminished end-volume at 30 cm H₂O at post-injury times of 24, 48, and 72 h; C. Total resistance of the respiratory system, with resistance increased most prominently at 24 h post-injury compared to control or 48 and 72 h of injury. D. Area under the Curve (AUC, the area enclosed by the inspiratory and expiratory loops of curves in Figure 3), showing decreased hysteresis post-contusion in consonance with decreased compliance seen with other maneuvers. One way ANOVA vs. control: * p < 0.05, ** p < 0.01, *** p < 0.001; One way ANOVA vs. 24 h injured animals # p < 0.05.

Figure 5. Dynamic surface tension lowering of resuspended large surfactant aggregates from mice with LC injury at 24 h post-injury

Centrifuged large surfactant aggregates were resuspended in 0.15 M NaCl + 2 mM CaCl₂ at a uniform phospholipid concentration of 2.5 mg/ml and examined for surface activity on a pulsating bubble surfactometer (20 cycles/min, 37°C, 50% area compression). Surface tension at minimum bubble radius (minimum surface tension) is shown as a function of time of bubble pulsation. Surface activity is reduced at all times of pulsation for mice at 24 h post-injury compared to uninjured controls. Data are Mean ± S.E.M. for n = 4.

Figure 6. Large surfactant aggregate (LG) content, phospholipase A₂ (PLA₂) concentration in cell-free BAL, and lyso-PC (LPC) content in large surfactant aggregates from uninjured and LC-injured mice

Large surfactant aggregates were obtained by centrifugation of cell-free BAL at 12,000 × g for 30 min, and the aggregate content was determined as a percentage of total BAL phospholipid content based on phosphate assay. Phospholipase A₂ concentration in cell-free BAL was measured with the PLAThe EnzChek® Phospholipase A₂ Assay Kit (Methods). Lyso-PC content in large surfactant aggregates was determined by thin layer chromatography. Data are Mean ± S.E.M. with n = 4 for LG and LPC, and n = 8 for PLA₂. *: p < 0.0001 compared with uninjured mice; #: p < 0.001 compared with uninjured mice; +: p < 0.01 compared with uninjured mice.