Recombinant tissue plasminogen activator enhances microparticle release from mouse brain-derived endothelial cells through plasmin

J Neurol Sci. 2016 Nov 15;370:187-195. doi: 10.1016/j.jns.2016.09.026. Epub 2016 Sep 16.

Abstract

Thrombolysis with recombinant tissue plasminogen activator (rt-PA) is currently the only approved pharmacological strategy for acute ischemic stroke. However, rt-PA exhibits vascular toxicity mainly due to endothelial damage. To investigate the mechanisms underlying rt-PA-induced endothelial alterations, we assessed the role of rt-PA in the generation of endothelial microparticles (EMPs), emerging biological markers and effectors of endothelial dysfunction. The mouse brain-derived endothelial cell line bEnd.3 was used. Cells were treated with rt-PA at 20, 40 or 80μg/ml for 15 or 24h, and EMPs were quantified in the culture media using Annexin-V staining coupled with flow cytometry. Rt-PA enhanced EMP release from bEnd.3 cells with a maximal increase at the 40μg/ml dose for 24h (+78% compared to controls). Using tranexamic acid and aprotinin we demonstrated that plasmin is responsible for rt-PA-induced EMP release. The p38 MAPK inhibitor SB203580 and the poly(ADP-ribose)polymerase (PARP) inhibitor PJ34 also reduced rt-PA-induced EMP production, suggesting that p38 MAPK and PARP are downstream intracellular effectors of rt-PA/plasmin. Rt-PA also altered through plasmin the morphology and the confluence of bEnd.3 cells. By contrast, these changes did not implicate p38 MAPK and PARP. This study demonstrates that rt-PA induces the production of microparticles by cerebral endothelial cells, through plasmin, p38 MAPK and PARP pathways. Determining the phenotype of these EMPs to clarify their role on the endothelium in ischemic conditions could thus be of particular interest.

Keywords: Brain endothelial cells; Microparticles; Microvesicles; Plasmin; Poly(ADP-ribose)polymerase (PARP); Recombinant tissue plasminogen activator (rt-PA); p38 MAPK.

MeSH terms

  • Animals
  • Brain / blood supply
  • Brain / drug effects
  • Brain / metabolism
  • Brain / pathology
  • Cell Line
  • Cell Survival / drug effects
  • Cell Survival / physiology
  • Cell-Derived Microparticles / drug effects*
  • Cell-Derived Microparticles / metabolism
  • Dose-Response Relationship, Drug
  • Endothelial Cells / drug effects*
  • Endothelial Cells / metabolism
  • Endothelial Cells / pathology
  • Fibrinolysin / metabolism*
  • Fibrinolytic Agents / pharmacology*
  • Mice
  • Plasminogen / metabolism
  • Poly(ADP-ribose) Polymerases / metabolism
  • Recombinant Proteins / pharmacology
  • Signal Transduction / drug effects
  • Time Factors
  • Tissue Plasminogen Activator / pharmacology*
  • p38 Mitogen-Activated Protein Kinases / antagonists & inhibitors
  • p38 Mitogen-Activated Protein Kinases / metabolism

Substances

  • Fibrinolytic Agents
  • Recombinant Proteins
  • Plasminogen
  • Poly(ADP-ribose) Polymerases
  • p38 Mitogen-Activated Protein Kinases
  • Tissue Plasminogen Activator
  • Fibrinolysin