title: N-Ethylmaleimide Sensitive Factor (NSF) Inhibition Prevents Vascular Instability following Gram-Positive Pulmonary Challenge.
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 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.
authors.affiliation: The Elmezzi Graduate School of Molecular Medicine, Manhasset, New York, United States of America.
authors.forename: Ji Young
authors.initials: JY
authors.lastname: Lee
authors.affiliation: The Center for Heart and Lung Research, The Feinstein Institute for Medical Research, Manhasset, New York, United States of America.
authors.forename: Helena M
authors.initials: HM
authors.lastname: Linge
authors.affiliation: The Center for Heart and Lung Research, The Feinstein Institute for Medical Research, Manhasset, New York, United States of America.
authors.forename: Kanta
authors.initials: K
authors.lastname: Ochani
authors.affiliation: The Center for Heart and Lung Research, The Feinstein Institute for Medical Research, Manhasset, New York, United States of America.
authors.forename: Ke
authors.initials: K
authors.lastname: Lin
authors.affiliation: The Elmezzi Graduate School of Molecular Medicine, Manhasset, New York, United States of America.|The Center for Heart and Lung Research, The Feinstein Institute for Medical Research, Manhasset, New York, United States of America.|Hofstra North Shore-LIJ Medical School, Hempstead, New York, United States of America.
authors.forename: Edmund J
authors.initials: EJ
authors.lastname: Miller
mesh_terms: D042683:Angiopoietin-1 / Q000378:metabolism; D000818:Animals; D001424:Bacterial Infections / Q000150:complications*; D001808:Blood Vessels / Q000473:pathology; D002460:Cell Line; D002473:Cell Wall / Q000187:drug effects; D003599:Cytoskeleton / Q000378:metabolism; D004195:Disease Models, Animal; D005089:Exocytosis; D006094:Gram-Positive Bacteria; D006801:Humans; D007249:Inflammation; D008070:Lipopolysaccharides / Q000737:chemistry; D008168:Lung / Q000382:microbiology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008833:Microcirculation; D052066:N-Ethylmaleimide-Sensitive Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D010100:Oxygen / Q000737:chemistry; D010457:Peptidoglycan / Q000737:chemistry; D010766:Phosphorylation; D011014:Pneumonia / Q000378:metabolism; D012128:Respiratory Distress Syndrome / Q000150:complications* / Q000382:microbiology; D018805:Sepsis / Q000378:metabolism; D013682:Teichoic Acids / Q000737:chemistry; D014652:Vascular Diseases / Q000378:metabolism / Q000517:prevention & control
chemical_list: D042683:Angiopoietin-1; D008070:Lipopolysaccharides; D010457:Peptidoglycan; D013682:Teichoic Acids; C009900:lipoteichoic acid; D052066:N-Ethylmaleimide-Sensitive Proteins; C498733:Nsf protein, mouse; D010100:Oxygen