title: Recombinant tissue plasminogen activator enhances microparticle release from mouse brain-derived endothelial cells through plasmin.
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.
authors: Equipe de recherche "Pharmacologie de la Circulation Cérébrale" EA4475, Faculté de Pharmacie de Paris, Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors: Marie
authors: M
authors: Garraud
authors: Equipe de recherche "Pharmacologie de la Circulation Cérébrale" EA4475, Faculté de Pharmacie de Paris, Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors: Kahina
authors: K
authors: Khacef
authors: INSERM, U970, Paris Cardiovascular Research Center, Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors: Anne-Clémence
authors: AC
authors: Vion
authors: Equipe de recherche "Pharmacologie de la Circulation Cérébrale" EA4475, Faculté de Pharmacie de Paris, Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors: Claire
authors: C
authors: Leconte
authors: INSERM, U970, Paris Cardiovascular Research Center, Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors: Min
authors: M
authors: Yin
authors: INSERM, U970, Paris Cardiovascular Research Center, Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors: Jean-Marie
authors: JM
authors: Renard
authors: Equipe de recherche "Pharmacologie de la Circulation Cérébrale" EA4475, Faculté de Pharmacie de Paris, Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors: Catherine
authors: C
authors: Marchand-Leroux
authors: INSERM, U970, Paris Cardiovascular Research Center, Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors: Chantal M
authors: CM
authors: Boulanger
authors: Equipe de recherche "Pharmacologie de la Circulation Cérébrale" EA4475, Faculté de Pharmacie de Paris, Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors: Isabelle
authors: I
authors: Margaill
authors: Equipe de recherche "Pharmacologie de la Circulation Cérébrale" EA4475, Faculté de Pharmacie de Paris, Université Paris Descartes, Sorbonne Paris Cité, Paris, France. Electronic address: virginie.berthat@parisdescartes.fr.
authors: Virginie
authors: V
authors: Beray-Berthat
mesh_terms: D000818:Animals; D001921:Brain / Q000098:blood supply / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D055252:Cell-Derived Microparticles / Q000187:drug effects* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005341:Fibrinolysin / Q000378:metabolism*; D005343:Fibrinolytic Agents / Q000494:pharmacology*; D051379:Mice; D010958:Plasminogen / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D010959:Tissue Plasminogen Activator / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D005343:Fibrinolytic Agents; D011994:Recombinant Proteins; D010958:Plasminogen; D011065:Poly(ADP-ribose) Polymerases; D048051:p38 Mitogen-Activated Protein Kinases; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin
keywords: Brain endothelial cells; Microparticles; Microvesicles; Plasmin; Poly(ADP-ribose)polymerase (PARP); Recombinant tissue plasminogen activator (rt-PA); p38 MAPK