Gene transfer of MRCKα rescues lipopolysaccharide-induced acute lung injury by restoring alveolar capillary barrier function

Abstract

Acute Lung Injury/Acute Respiratory Distress Syndrome (ALI/ARDS) is characterized by alveolar edema accumulation with reduced alveolar fluid clearance (AFC), alveolar-capillary barrier disruption, and substantial inflammation, all leading to acute respiratory failure. Enhancing AFC has long been considered one of the primary therapeutic goals in gene therapy treatments for ARDS. We previously showed that electroporation-mediated gene delivery of the Na⁺, K⁺-ATPase β1 subunit not only increased AFC, but also restored alveolar barrier function through upregulation of tight junction proteins, leading to treatment of LPS-induced ALI in mice. We identified MRCKα as an interaction partner of β1 which mediates this upregulation in cultured alveolar epithelial cells. In this study, we investigate whether electroporation-mediated gene transfer of MRCKα to the lungs can attenuate LPS-induced acute lung injury in vivo. Compared to mice that received a non-expressing plasmid, those receiving the MRCKα plasmid showed attenuated LPS-increased pulmonary edema and lung leakage, restored tight junction protein expression, and improved overall outcomes. Interestingly, gene transfer of MRCKα did not alter AFC rates. Studies using both cultured microvascular endothelial cells and mice suggest that β1 and MRCKα upregulate junctional complexes in both alveolar epithelial and capillary endothelial cells, and that one or both barriers may be positively affected by our approach. Our data support a model of treatment for ALI/ARDS in which improvement of alveolar-capillary barrier function alone may be of more benefit than improvement of alveolar fluid clearance.

Figure 1

Overexpression MRCKα increases tight junction protein expression in healthy mouse lungs. Plasmids (100 µg each) expressing either no insert (pcDNA3), the β1 subunit of the Na⁺,K⁺-ATPase (β1), MRCKα, or β1 and MRCKα were delivered in 50 µl to the lungs of C75B6 mice (n = 3) by aspiration followed immediately by electroporation (8 pulses of 10 ms duration each and 200 V/cm). Two days later, lungs were perfused with PBS and lysates were prepared for analysis by Western Blot (A). Levels of expression were normalized to GAPDH as a loading control and the relative expression of ZO-1 (white bars) and Occludin (grey bars) are shown as mean ± SEM (B). All experiments were carried out three times and a representative experiment is shown. One-way ANOVA with post-hoc Tukey’s multiple comparisons was used for statistical analysis; a, p < 0.01 compared to naïve; b, p < 0.05 compared to naïve; c, p < 0.001 compared to naïve; d, p < 0.05 compared to pcDNA3; e, p < 0.01 compared to pcDNA3.

Figure 2

Experimental timeline for treatment of lung injury. Lung injury was established in mice by 5 mg/kg LPS administered by aspiration and 1 day later, plasmids (either β1, MRCKα, or a combination of the two; 100 µg in 50 µl PBS) were aspirated into the lungs and electroporated using electrodes placed on either side of the chest. Two days after gene delivery, lung injury was assessed.

Figure 3

Overexpression of MRCKα restores ZO-1 and occludin expression in previously injured mouse lungs. Lung injury was established in C57B6 mice (n = 6–8) by aspiration of LPS (5 mg/kg) and 1 day later plasmids (100 µg each) expressing either no insert (pcDNA3), the β1 subunit of the Na⁺,K⁺-ATPase (β1), MRCKα, or β1 and MRCKα were delivered in 50 µl to the lungs by aspiration followed immediately by electroporation (8 pulses of 10 ms duration each and 200 V/cm). Two days later (3 days after LPS administration), lungs were perfused with PBS and lysates were prepared for analysis by Western Blot (A). Levels of expression were normalized to GAPDH as a loading control and the relative expression of Occludin (B) and ZO-1 (C) are shown as mean ± SEM. All experiments were carried out three times and representative experiments are shown. One-way ANOVA with post-hoc Tukey’s multiple comparisons was used for statistical analysis; a, p < 0.05 compared to naïve; b, p < 0.05 compared to pcDNA3.

Figure 4

Overexpression of MRCKα or the Na⁺,K⁺-ATPase β1 subunit in microvascular endothelial cells attenuates LPS-induced reduction of VE-cadherin expression. Human MVECs grown on coverslips were transfected with plasmids (2 µg/well) expressing either no insert (pcDNA3), MRCKα, the β1 subunit of the Na⁺,K⁺-ATPase (β1), or β1 and MRCKα. Forty-eight hours later, cells were treated with nothing (Naïve) or LPS (1 µg/ml) for 5 h prior to fixation and immunofluorescent staining for VE-cadherin. The experiment was carried out on triplicate coverslips in three different experiments and representative images from two different wells are shown.

Figure 5

Electroporation-mediated gene transfer of MRCKα to mice with existing lung injury attenuates LPS-induced reduction in VE-cadherin. Lung injury was established in C57B6 mice (n = 6–12) by aspiration of LPS (5 mg/kg) and 1 day later plasmids (100 µg each) expressing either no insert (pcDNA3), the β1 subunit of the Na⁺,K⁺-ATPase (β1), MRCKα, or β1 and MRCKα were delivered in 50 µl to the lungs by aspiration followed immediately by electroporation (8 pulses of 10 ms duration each and 200 V/cm). Two days later (3 days after LPS administration), lungs were perfused with PBS and lysates were used for Western blots of VE-cadherin expression (A). Levels of expression were normalized to GAPDH as a loading control and the relative expression of VE-cadherin (B) is shown as mean ± SEM. All experiments were carried out three times and representative experiments are shown. One-way ANOVA with post-hoc Tukey’s multiple comparisons was used for statistical analysis; a, p < 0.05 compared to LPS only; b, p < 0.01 compared to LPS only; c, p < 0.05 compared to pcDNA3; and d, p < 0.01 compared to pcDNA3.

Figure 6

Electroporation mediated gene transfer of MRCKα attenuates lung leakage in lungs of mice previously injured with LPS. Lung injury was established in C57B6 mice (n = 9–11) by aspiration of LPS (5 mg/kg) and 1 day later plasmids (100 µg each) expressing either no insert (pcDNA3), the β1 subunit of the Na⁺, K⁺-ATPase (β1), MRCKα, or β1 and MRCKα were delivered in 50 µl to the lungs by aspiration followed immediately by electroporation (8 pulses of 10 ms duration each and 200 V/cm). Forty-seven hours later, Evans Blue Dye (30 mg/kg) was administered by tail vein injection and one hour later, lungs were perfused with PBS and harvested for Evans Blue Dye extraction. Lung permeability was evaluated by quantifying the absorbance of extracted Evans Blue Dye and shown as mean ± SEM. All experiments were carried out three times and a representative experiment is shown. One-way ANOVA with post-hoc Tukey’s multiple comparisons was used for statistical analysis; a, p ≤ 0.0001 compared to naïve; b, p < 0.05 compared to LPS; c, p < 0.01 compared to LPS; d, p < 0.05 compared to pcDNA3; e, p < 0.01 compared to pcDNA3.

Figure 7

Electroporation mediated gene transfer of MRCKα attenuates lung edema fluid accumulation in previously injured lungs. Lung injury was established in C57B6 mice (n = 5–8) by aspiration of LPS (5 mg/kg) and 1 day later plasmids (100 µg each) expressing either no insert (pcDNA3), the β1 subunit of the Na⁺,K⁺-ATPase (β1), MRCKα, or β1 and MRCKα were delivered in 50 µl to the lungs by aspiration followed immediately by electroporation (8 pulses of 10 ms duration each and 200 V/cm). Two days later (3 days after LPS administration), wet to dry ratios were determined as a measure of pulmonary edema fluid and shown as mean ± SEM. All experiments were carried out three times and a representative experiment is shown. One-way ANOVA with post-hoc Tukey’s multiple comparisons was used for statistical analysis; a, p < 0.0001 compared to naïve; b, p < 0.01 compared to naïve; c, p < 0.05 compared to LPS; d, p < 0.05 compared to pcDNA3; e, p < 0.01 compared to pcDNA3.

Figure 8

Gene delivery of MRCKα to mice with existing lung injury attenuates inflammatory cell infiltration. Lung injury was established in C57B6 mice (n = 8–10) by aspiration of LPS (5 mg/kg) and 1 day later plasmids (100 µg each) expressing either no insert (pcDNA3), the β1 subunit of the Na⁺,K⁺-ATPase (β1), MRCKα, or β1 and MRCKα were delivered in 50 µl to the lungs by aspiration followed immediately by electroporation (8 pulses of 10 ms duration each and 200 V/cm). Naïve mice (n = 5) received no LPS or DNA. Two days later (3 days after LPS administration), lungs were lavaged with PBS and BAL fluid was collected and analyzed for cellularity by cytospin followed by Diff-quik staining (A). All experiments were carried out three times and a representative experiment is shown. Total cells were quantified in the BAL fluid and shown as mean ± SEM (B). One-way ANOVA with post-hoc Tukey’s multiple comparisons was used for statistical analysis; a, p < 0.0001 compared to naïve; b, p < 0.001 compared to naïve; c, p < 0.05 compared to LPS; d, p < 0.01 compared to LPS; e, p < 0.05 compared to pcDNA3; f, p < 0.01 compared to pcDNA3. The number of PMNs in the BAL fluid were also quantified and shown as mean ± SEM (C). One-way ANOVA with post-hoc Tukey’s multiple comparisons was used for statistical analysis; a, p < 0.0001 compared to naïve; b, p < 0.01 compared to naïve; c, p < 0.05 compared to LPS; d, p < 0.01 compared to LPS; e, p < 0.05 compared to pcDNA3;f, p < 0.01 compared to pcDNA3.

Figure 9

MRCKα gene transfer reduces BAL protein levels in previously injured mice. Lung injury was established in C57B6 mice (n = 8–9) by aspiration of LPS (5 mg/kg) and 1 day later plasmids (100 µg each) expressing either no insert (pcDNA3), the β1 subunit of the Na⁺,K⁺-ATPase (β1), MRCKα, or β1 and MRCKα were delivered in 50 µl to the lungs by aspiration followed immediately by electroporation (8 pulses of 10 ms duration each and 200 V/cm). Naïve mice (n = 5) received no LPS or DNA. Two days later (3 days after LPS administration), lungs were lavaged with PBS and BAL fluid was collected and analyzed for total protein content, shown as mean ± SEM (A). All experiments were carried out three times and a representative experiment is shown. One-way ANOVA with post-hoc Tukey’s multiple comparisons was used for statistical analysis; a, p < 0.0001 compared to naïve; b, p < 0.01 compared to naïve; c, p < 0.05 compared to LPS; d, p < 0.001 compared to LPS; e, p < 0.05 compared to pcDNA3; f, p < 0.001 compared to pcDNA3. The concentration of albumin in the BAL fluid was quantified by ELISA and shown as mean ± SEM (B). One-way ANOVA with post-hoc Tukey’s multiple comparisons was used for statistical analysis; a, p < 0.001 compared to naïve; b, p < 0.001 compared to LPS; c, p < 0.0001 compared to pcDNA3; d, p < 0.01 compared to LPS.

Figure 10

Electroporation-mediated gene transfer of MRCKα improves overall histology of mice with pre-existing LPS-induced lung injury. Lung injury was established in C57B6 mice (n = 6–8) by aspiration of LPS (5 mg/kg) and 1 day later plasmids (100 µg each) expressing either no insert (pcDNA3), the β1 subunit of the Na⁺,K⁺-ATPase (β1), MRCKα, or β1 and MRCKα were delivered in 50 µl to the lungs by aspiration followed immediately by electroporation (8 pulses of 10 ms duration each and 200 V/cm). Naïve mice (n = 7) received no LPS or DNA. Two days later (3 days after LPS administration), lungs were inflated to 20 cm H₂O with 10% buffered formalin and processed for paraffin-embedding, sectioning, and hematoxylin and eosin staining. Sections from 5 representative animals are shown at ×50 (A) and ×400 (B) magnification. All experiments were carried out three times and a representative experiment is shown. Scale bar is 400 µm (A) and 50 µm (B).

Figure 11

Electroporation mediated gene transfer of MRCKα has no effect on rates of alveolar fluid clearance. Plasmids (100 µg each) expressing either no insert (pcDNA3), the β1 subunit of the Na⁺,K⁺-ATPase (β1), MRCKα, or β1 and MRCKα were delivered in 50 µl to the lungs of C75B6 mice (n = 6–7) by aspiration followed immediately by electroporation (8 pulses of 10 ms duration each and 200 V/cm). Naïve mice received no DNA. Two days later, alveolar fluid clearance was measured in living mice and calculated based on the change in concentration of Evans Blue Dye-labeled albumin in an isosmolar (324 mOsm) instillate placed into the alveolar space and mechanically ventilated over a 30 min period. Procaterol (10⁻⁸ mol/L) was administered in the instillate and used as the positive control in a set of naïve mice. Rates of alveolar fluid clearance are shown as mean ± SEM. All experiments were carried out three times and a representative experiment is shown. One-way ANOVA with post-hoc Tukey’s multiple comparisons was used for statistical analysis; a, p < 0.01 compared to naïve; b, p < 0.001 compared to pcDNA3; c, p < 0.01 compared to MRCKα; d, p < 0.05 compared to MRCKα.