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Mitogen activated protein kinase activated protein kinase 2 regulates actin polymerization and vascular leak in ventilator associated lung injury.

Damarla M, Hasan E, Boueiz A, Le A, Pae HH, Montouchet C, Kolb T, Simms T, Myers A, Kayyali US, Gaestel M, Peng X, Reddy SP, Damico R, Hassoun PM - PLoS ONE (2009)

Bottom Line: However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability.Finally, MK2(-/-) mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HV(T) MV.Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury. Wild type and MK2(-/-) mice received mechanical ventilation with high (20 ml/kg) or low (7 ml/kg) tidal volumes up to 4 hrs, after which lungs were harvested for immunohistochemistry, immunoblotting and lung permeability assays. High tidal volume mechanical ventilation resulted in significant phosphorylation of p38 MAP kinase, MK2, HSP25, actin polymerization, and an increase in pulmonary vascular permeability in wild type mice as compared to spontaneous breathing or low tidal volume mechanical ventilation. However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability. Finally, MK2(-/-) mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HV(T) MV. Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling.

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High tidal volume mechanical ventilation induces actin polymerization.Wild type or MK2−/− mice were randomized to spontaneous breathing or MV at LVT (7 ml/kg) and HVT (20 ml/kg) for 240 minutes (4 hrs). Lung tissue sections were prepared and stained for G-actin, F-actin and nuclei as described in the Methods section. A. Representative images (100× images and inset 20×) from wild type spontaneously breathing (Control) mice or those exposed to MV at LVT or HVT for 4 hrs. B. Representative images (100× images and inset 20×) from wild type mice pretreated with the p38 MAP kinase inhibitor SB203580, MK2 inhibitor KKKALNRQLGVAA or MK2−/− mice exposed to MV at HVT for 4 hrs. Images from MK2−/− mice exposed to spontaneous breathing conditions (not shown) are not different from those from mice exposed to HVT MV. C. Relative intensity of F-actin and G-actin was assessed from greater than 60 random images at low power (20× magnification) per condition and graphed as F-actin to G-actin ratio to represent actin polymerization. The F-actin to G-actin ratio after MV at HVT was significantly higher compared to MV at LVT or pre-treatment with SB203580, KKKALNRQLGVAA (3.02±1.0, 1.55±0.69, 1.56±0.19 and 0.97±0.34, respectively). Control represents spontaneously breathing conditions. MK2-deficient mice have a lower F-actin to G-actin ratio at baseline and are unable to polymerize actin in response to MV at HVT (0.63±0.29 at control conditions and 0.67±0.28 after MV at HVT. * P<0.05 (vehicle vs all others). There was no statistical difference in the F-actin to G-actin ratio between MK2−/− mice under control conditions and those exposed to 4 hr of HVT MV. N = 3–4 mice per group.
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pone-0004600-g004: High tidal volume mechanical ventilation induces actin polymerization.Wild type or MK2−/− mice were randomized to spontaneous breathing or MV at LVT (7 ml/kg) and HVT (20 ml/kg) for 240 minutes (4 hrs). Lung tissue sections were prepared and stained for G-actin, F-actin and nuclei as described in the Methods section. A. Representative images (100× images and inset 20×) from wild type spontaneously breathing (Control) mice or those exposed to MV at LVT or HVT for 4 hrs. B. Representative images (100× images and inset 20×) from wild type mice pretreated with the p38 MAP kinase inhibitor SB203580, MK2 inhibitor KKKALNRQLGVAA or MK2−/− mice exposed to MV at HVT for 4 hrs. Images from MK2−/− mice exposed to spontaneous breathing conditions (not shown) are not different from those from mice exposed to HVT MV. C. Relative intensity of F-actin and G-actin was assessed from greater than 60 random images at low power (20× magnification) per condition and graphed as F-actin to G-actin ratio to represent actin polymerization. The F-actin to G-actin ratio after MV at HVT was significantly higher compared to MV at LVT or pre-treatment with SB203580, KKKALNRQLGVAA (3.02±1.0, 1.55±0.69, 1.56±0.19 and 0.97±0.34, respectively). Control represents spontaneously breathing conditions. MK2-deficient mice have a lower F-actin to G-actin ratio at baseline and are unable to polymerize actin in response to MV at HVT (0.63±0.29 at control conditions and 0.67±0.28 after MV at HVT. * P<0.05 (vehicle vs all others). There was no statistical difference in the F-actin to G-actin ratio between MK2−/− mice under control conditions and those exposed to 4 hr of HVT MV. N = 3–4 mice per group.

Mentions: As our laboratory has demonstrated increased stress fiber formation with MK2 activation and HSP27 phosphorylation in vitro [9], we next explored the potential effect of activation of the p38 MAP kinase-MK2-HSP25 signaling pathway on actin polymerization in response to HVT MV. Lung tissue sections obtained from mice exposed to MV were probed for F-actin and G-actin, as detailed in Materials and Methods. As demonstrated in Figure 4, there was significant actin polymerization, as evidenced by an increase in the F-actin to G-actin ratio, in response to MV at HVT but not LVT. In addition, pretreatment of mice with the p38 MAP kinase inhibitor SB203580 or the MK2 inhibitory peptide KKKALNRQLGVAA significantly abrogated the increase in F-actin to G-actin ratio. Finally, MK2−/− mice displayed a low F-actin to G-actin ratio at baseline (compared to wild type counterparts) and failed to increase this ratio in response to HVT MV. Taken together, these results indicate that MV at HVT (but not LVT) results in actin polymerization which is dependent on activation of p38 MAP kinase and MK2 with subsequent HSP25 phosphorylation.


Mitogen activated protein kinase activated protein kinase 2 regulates actin polymerization and vascular leak in ventilator associated lung injury.

Damarla M, Hasan E, Boueiz A, Le A, Pae HH, Montouchet C, Kolb T, Simms T, Myers A, Kayyali US, Gaestel M, Peng X, Reddy SP, Damico R, Hassoun PM - PLoS ONE (2009)

High tidal volume mechanical ventilation induces actin polymerization.Wild type or MK2−/− mice were randomized to spontaneous breathing or MV at LVT (7 ml/kg) and HVT (20 ml/kg) for 240 minutes (4 hrs). Lung tissue sections were prepared and stained for G-actin, F-actin and nuclei as described in the Methods section. A. Representative images (100× images and inset 20×) from wild type spontaneously breathing (Control) mice or those exposed to MV at LVT or HVT for 4 hrs. B. Representative images (100× images and inset 20×) from wild type mice pretreated with the p38 MAP kinase inhibitor SB203580, MK2 inhibitor KKKALNRQLGVAA or MK2−/− mice exposed to MV at HVT for 4 hrs. Images from MK2−/− mice exposed to spontaneous breathing conditions (not shown) are not different from those from mice exposed to HVT MV. C. Relative intensity of F-actin and G-actin was assessed from greater than 60 random images at low power (20× magnification) per condition and graphed as F-actin to G-actin ratio to represent actin polymerization. The F-actin to G-actin ratio after MV at HVT was significantly higher compared to MV at LVT or pre-treatment with SB203580, KKKALNRQLGVAA (3.02±1.0, 1.55±0.69, 1.56±0.19 and 0.97±0.34, respectively). Control represents spontaneously breathing conditions. MK2-deficient mice have a lower F-actin to G-actin ratio at baseline and are unable to polymerize actin in response to MV at HVT (0.63±0.29 at control conditions and 0.67±0.28 after MV at HVT. * P<0.05 (vehicle vs all others). There was no statistical difference in the F-actin to G-actin ratio between MK2−/− mice under control conditions and those exposed to 4 hr of HVT MV. N = 3–4 mice per group.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2643011&req=5

pone-0004600-g004: High tidal volume mechanical ventilation induces actin polymerization.Wild type or MK2−/− mice were randomized to spontaneous breathing or MV at LVT (7 ml/kg) and HVT (20 ml/kg) for 240 minutes (4 hrs). Lung tissue sections were prepared and stained for G-actin, F-actin and nuclei as described in the Methods section. A. Representative images (100× images and inset 20×) from wild type spontaneously breathing (Control) mice or those exposed to MV at LVT or HVT for 4 hrs. B. Representative images (100× images and inset 20×) from wild type mice pretreated with the p38 MAP kinase inhibitor SB203580, MK2 inhibitor KKKALNRQLGVAA or MK2−/− mice exposed to MV at HVT for 4 hrs. Images from MK2−/− mice exposed to spontaneous breathing conditions (not shown) are not different from those from mice exposed to HVT MV. C. Relative intensity of F-actin and G-actin was assessed from greater than 60 random images at low power (20× magnification) per condition and graphed as F-actin to G-actin ratio to represent actin polymerization. The F-actin to G-actin ratio after MV at HVT was significantly higher compared to MV at LVT or pre-treatment with SB203580, KKKALNRQLGVAA (3.02±1.0, 1.55±0.69, 1.56±0.19 and 0.97±0.34, respectively). Control represents spontaneously breathing conditions. MK2-deficient mice have a lower F-actin to G-actin ratio at baseline and are unable to polymerize actin in response to MV at HVT (0.63±0.29 at control conditions and 0.67±0.28 after MV at HVT. * P<0.05 (vehicle vs all others). There was no statistical difference in the F-actin to G-actin ratio between MK2−/− mice under control conditions and those exposed to 4 hr of HVT MV. N = 3–4 mice per group.
Mentions: As our laboratory has demonstrated increased stress fiber formation with MK2 activation and HSP27 phosphorylation in vitro [9], we next explored the potential effect of activation of the p38 MAP kinase-MK2-HSP25 signaling pathway on actin polymerization in response to HVT MV. Lung tissue sections obtained from mice exposed to MV were probed for F-actin and G-actin, as detailed in Materials and Methods. As demonstrated in Figure 4, there was significant actin polymerization, as evidenced by an increase in the F-actin to G-actin ratio, in response to MV at HVT but not LVT. In addition, pretreatment of mice with the p38 MAP kinase inhibitor SB203580 or the MK2 inhibitory peptide KKKALNRQLGVAA significantly abrogated the increase in F-actin to G-actin ratio. Finally, MK2−/− mice displayed a low F-actin to G-actin ratio at baseline (compared to wild type counterparts) and failed to increase this ratio in response to HVT MV. Taken together, these results indicate that MV at HVT (but not LVT) results in actin polymerization which is dependent on activation of p38 MAP kinase and MK2 with subsequent HSP25 phosphorylation.

Bottom Line: However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability.Finally, MK2(-/-) mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HV(T) MV.Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury. Wild type and MK2(-/-) mice received mechanical ventilation with high (20 ml/kg) or low (7 ml/kg) tidal volumes up to 4 hrs, after which lungs were harvested for immunohistochemistry, immunoblotting and lung permeability assays. High tidal volume mechanical ventilation resulted in significant phosphorylation of p38 MAP kinase, MK2, HSP25, actin polymerization, and an increase in pulmonary vascular permeability in wild type mice as compared to spontaneous breathing or low tidal volume mechanical ventilation. However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability. Finally, MK2(-/-) mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HV(T) MV. Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling.

Show MeSH
Related in: MedlinePlus