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N-Ethylmaleimide Sensitive Factor (NSF) Inhibition Prevents Vascular Instability following Gram-Positive Pulmonary Challenge

Lee, Ji Young; Linge, Helena M LU ; Ochani, Kanta; Lin, Ke and Miller, Edmund J (2016) In PLoS ONE 11(6).
Abstract

BACKGROUND: The Acute Respiratory Distress Syndrome (ARDS), remains a significant source of morbidity and mortality in critically ill patients. Pneumonia and sepsis are leading causes of ARDS, the pathophysiology of which includes increased pulmonary microvascular permeability and hemodynamic instability resulting in organ dysfunction. We hypothesized that N-ethylmaleimide sensitive factor (NSF) regulates exocytosis of inflammatory mediators, such as Angiopoietin-2 (Ang-2), and cytoskeletal stability by modulating myosin light chain (MLC) phosphorylation. Therefore, we challenged pulmonary cells, in vivo and in vitro, with Gram Positive bacterial cell wall components, lipoteichoic acid (LTA), and peptidoglycan (PGN) and examined the... (More)

BACKGROUND: The Acute Respiratory Distress Syndrome (ARDS), remains a significant source of morbidity and mortality in critically ill patients. Pneumonia and sepsis are leading causes of ARDS, the pathophysiology of which includes increased pulmonary microvascular permeability and hemodynamic instability resulting in organ dysfunction. We hypothesized that N-ethylmaleimide sensitive factor (NSF) regulates exocytosis of inflammatory mediators, such as Angiopoietin-2 (Ang-2), and cytoskeletal stability by modulating myosin light chain (MLC) phosphorylation. Therefore, we challenged pulmonary cells, in vivo and in vitro, with Gram Positive bacterial cell wall components, lipoteichoic acid (LTA), and peptidoglycan (PGN) and examined the effects of NSF inhibition.

METHODS: Mice were pre-treated with an inhibitor of NSF, TAT-NSF700 (to prevent Ang-2 release). After 30min, LTA and PGN (or saline alone) were instilled intratracheally. Pulse oximetry was assessed in awake mice prior to, and 6 hour post instillation. Post mortem, tissues were collected for studies of inflammation and Ang-2. In vitro, pulmonary endothelial cells were assessed for their responses to LTA and PGN.

RESULTS: Pulmonary challenge induced signs of airspace and systemic inflammation such as changes in neutrophil counts and protein concentration in bronchoalveolar lavage fluid and tissue Ang-2 concentration, and decreased physiological parameters including oxygen saturation and pulse distention. TAT-NSF700 pre-treatment reduced LTA-PGN induced changes in lung tissue Ang-2, oxygen saturation and pulse distention. In vitro, LTA-PGN induced a rapid (<2 min) release of Ang-2, which was significantly attenuated by TAT-NSF700 or anti TLR2 antibody. Furthermore, TAT-NSF700 reduced LTA-PGN-induced MLC phosphorylation at low concentrations of 1-10 nM.

CONCLUSIONS: TAT-NSF700 decreased Ang-2 release, improved oxygen saturation and pulse distention following pulmonary challenge by inhibiting MLC phosphorylation, an important component of endothelial cell retraction. The data suggest that inhibition of NSF in pneumonia and sepsis may be beneficial to prevent the pulmonary microvascular and hemodynamic instability associated with ARDS.

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published
keywords
Journal Article
in
PLoS ONE
volume
11
issue
6
publisher
Public Library of Science
external identifiers
  • scopus:84977495291
ISSN
1932-6203
DOI
10.1371/journal.pone.0157837
language
English
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no
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b8910978-afdc-434a-8bdb-368e1e1e1978
date added to LUP
2016-10-20 11:56:46
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2017-07-02 04:55:14
@article{b8910978-afdc-434a-8bdb-368e1e1e1978,
  abstract     = {<p>BACKGROUND: The Acute Respiratory Distress Syndrome (ARDS), remains a significant source of morbidity and mortality in critically ill patients. Pneumonia and sepsis are leading causes of ARDS, the pathophysiology of which includes increased pulmonary microvascular permeability and hemodynamic instability resulting in organ dysfunction. We hypothesized that N-ethylmaleimide sensitive factor (NSF) regulates exocytosis of inflammatory mediators, such as Angiopoietin-2 (Ang-2), and cytoskeletal stability by modulating myosin light chain (MLC) phosphorylation. Therefore, we challenged pulmonary cells, in vivo and in vitro, with Gram Positive bacterial cell wall components, lipoteichoic acid (LTA), and peptidoglycan (PGN) and examined the effects of NSF inhibition.</p><p>METHODS: Mice were pre-treated with an inhibitor of NSF, TAT-NSF700 (to prevent Ang-2 release). After 30min, LTA and PGN (or saline alone) were instilled intratracheally. Pulse oximetry was assessed in awake mice prior to, and 6 hour post instillation. Post mortem, tissues were collected for studies of inflammation and Ang-2. In vitro, pulmonary endothelial cells were assessed for their responses to LTA and PGN.</p><p>RESULTS: Pulmonary challenge induced signs of airspace and systemic inflammation such as changes in neutrophil counts and protein concentration in bronchoalveolar lavage fluid and tissue Ang-2 concentration, and decreased physiological parameters including oxygen saturation and pulse distention. TAT-NSF700 pre-treatment reduced LTA-PGN induced changes in lung tissue Ang-2, oxygen saturation and pulse distention. In vitro, LTA-PGN induced a rapid (&lt;2 min) release of Ang-2, which was significantly attenuated by TAT-NSF700 or anti TLR2 antibody. Furthermore, TAT-NSF700 reduced LTA-PGN-induced MLC phosphorylation at low concentrations of 1-10 nM.</p><p>CONCLUSIONS: TAT-NSF700 decreased Ang-2 release, improved oxygen saturation and pulse distention following pulmonary challenge by inhibiting MLC phosphorylation, an important component of endothelial cell retraction. The data suggest that inhibition of NSF in pneumonia and sepsis may be beneficial to prevent the pulmonary microvascular and hemodynamic instability associated with ARDS.</p>},
  articleno    = {e0157837},
  author       = {Lee, Ji Young and Linge, Helena M and Ochani, Kanta and Lin, Ke and Miller, Edmund J},
  issn         = {1932-6203},
  keyword      = {Journal Article},
  language     = {eng},
  number       = {6},
  publisher    = {Public Library of Science},
  series       = {PLoS ONE},
  title        = {N-Ethylmaleimide Sensitive Factor (NSF) Inhibition Prevents Vascular Instability following Gram-Positive Pulmonary Challenge},
  url          = {http://dx.doi.org/10.1371/journal.pone.0157837},
  volume       = {11},
  year         = {2016},
}