title: α-Enolase Causes Proinflammatory Activation of Pulmonary Microvascular Endothelial Cells and Primes Neutrophils Through Plasmin Activation of Protease-Activated Receptor 2.
abstract: Proinflammatory activation of vascular endothelium leading to increased surface expression of adhesion molecules and neutrophil (PMN) sequestration and subsequent activation is paramount in the development of acute lung injury and organ injury in injured patients. We hypothesize that α-enolase, which accumulates in injured patients, primes PMNs and causes proinflammatory activation of endothelial cells leading to PMN-mediated cytotoxicity.
  
  
  METHODS
  Proteomic analyses of field plasma samples from injured versus healthy patients were used for protein identification. Human pulmonary microvascular endothelial cells (HMVECs) were incubated with α-enolase or thrombin, and intercellular adhesion molecule-1 surface expression was measured by flow cytometry. A two-event in vitro model of PMN cytotoxicity HMVECs activated with α-enolase, thrombin, or buffer was used as targets for lysophosphatidylcholine-primed or buffer-treated PMNs. The PMN priming activity of α-enolase was completed, and lysates from both PMNs and HMVECs were immunoblotted for protease-activated receptor 1 (PAR-1) and PAR-2 and coprecipitation of α-enolase with PAR-2 and plasminogen/plasmin.
  
  
  RESULTS
  α-Enolase increased 10.8-fold in injured patients (P < 0.05). Thrombin and α-enolase significantly increased intercellular adhesion molecule-1 surface expression on HMVECs, which was inhibited by antiproteases, induced PMN adherence, and served as the first event in the two-event model of PMN cytotoxicity. α-Enolase coprecipitated with PAR-2 and plasminogen/plasmin on HMVECs and PMNs and induced PMN priming, which was inhibited by tranexamic acid, and enzymatic activity was not required.
  
  
  CONCLUSIONS
  α-Enolase increases after injury and may activate pulmonary endothelial cells and prime PMNs through plasmin activity and PAR-2 activation. Such proinflammatory endothelial activation may predispose to PMN-mediated organ injury.
authors.affiliation: *Research Laboratory Bonfils Blood Center and †School of Medicine, University of Colorado Denver; Departments of ‡Surgery, §Pediatrics, and ∥Cell Biology, School of Medicine, University of Colorado Denver, Aurora; and ¶School of Public Health, University of Colorado; and **Department of Surgery Denver Health Medical Center, Denver, Colorado.
authors.forename: Ashley
authors.initials: A
authors.lastname: Bock
authors.forename: Nicole
authors.initials: N
authors.lastname: Tucker
authors.forename: Marguerite R
authors.initials: MR
authors.lastname: Kelher
authors.forename: Samina Y
authors.initials: SY
authors.lastname: Khan
authors.forename: Eduardo
authors.initials: E
authors.lastname: Gonzalez
authors.forename: Max
authors.initials: M
authors.lastname: Wohlauer
authors.forename: Kirk
authors.initials: K
authors.lastname: Hansen
authors.forename: Monika
authors.initials: M
authors.lastname: Dzieciatkowska
authors.forename: Angels
authors.initials: A
authors.lastname: Sauaia
authors.forename: Anirban
authors.initials: A
authors.lastname: Banerjee
authors.forename: Ernest E
authors.initials: EE
authors.lastname: Moore
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Silliman
mesh_terms: D055371:Acute Lung Injury / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D007249:Inflammation; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D008168:Lung / Q000098:blood supply*; D008833:Microcirculation; D009504:Neutrophils / Q000378:metabolism*; D010751:Phosphopyruvate Hydratase / Q000378:metabolism*; D010958:Plasminogen / Q000378:metabolism*; D040901:Proteomics; D044464:Receptor, PAR-2 / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism; D014148:Tranexamic Acid / Q000378:metabolism; D014949:Wounds, Nonpenetrating / Q000097:blood
chemical_list: C573420:ICAM1 protein, human; D044464:Receptor, PAR-2; D018799:Intercellular Adhesion Molecule-1; D014148:Tranexamic Acid; D010958:Plasminogen; D013917:Thrombin; D010751:Phosphopyruvate Hydratase