title: Protease activated receptor-1 mediates cytotoxicity during ischemia using in vivo and in vitro models.
abstract: Protease activated receptors (PARs) populate neurons and astrocytes in the brain. The serine protease thrombin, which activates PAR-1 during the first hours after stroke, appears to be associated with the cytotoxicity. Thrombin antagonists and PAR-1 inhibitors have been correlated with reduced cell death and behavioral protection after stroke, but no data yet support a mechanistic link between PAR-1 action and benefit. We sought to establish the essential role of PAR-1 in mediating ischemic damage. Using a short hairpin mRNA packaged with green fluorescent protein in a lentivirus vector, we knocked downPAR-1 in the medial caudate nucleus prior to rat middle cerebral artery occlusion (MCAo) and in rat neurons prior to oxygen-glucose deprivation. We also compared aged PAR-1 knockout mice with aged PAR-3, PAR-4 mice and young wild-type mice in a standard MCAo model. Silencing PAR-1 significantly reduced neurological deficits, reduced endothelial barrier leakage, and decreased neuronal degeneration in vivo during MCAo. PAR-1 knock-down in the ischemic medial caudate allowed cells to survive the ischemic injury; infected cells were negative for terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling (TUNEL) and c-Fos injury markers. Primary cultured neurons infected with PAR-1 short hairpin ribonucleic acid (shRNA) showed increased neuroprotection during hypoxic/aglycemic conditions with or without added thrombin. The aged PAR-1 knockout mice showed decreased infarction and vascular disruption compared to aged controls or young wild types. We demonstrated an essential role for PAR-1 during ischemia. Silencing or removing PAR-1 significantly protected neurons and astrocytes. Further development of agents that act at PAR-1 or its downstream pathways could yield powerful stroke therapy.
authors.affiliation: Department of Neurology, Cedars-Sinai Medical Center, Los Angeles 90048, USA.
authors.forename: P S
authors.initials: PS
authors.lastname: Rajput
authors.affiliation: Department of Neurology, Cedars-Sinai Medical Center, Los Angeles 90048, USA. Electronic address: lydenp@cshs.org.
authors.forename: P D
authors.initials: PD
authors.lastname: Lyden
authors.affiliation: Department of Neurology, Cedars-Sinai Medical Center, Los Angeles 90048, USA; McKinsey & Company, Beijing, China.
authors.forename: B
authors.initials: B
authors.lastname: Chen
authors.affiliation: Department of Neurology, Cedars-Sinai Medical Center, Los Angeles 90048, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Lamb
authors.affiliation: Department of Neurology, Cedars-Sinai Medical Center, Los Angeles 90048, USA.
authors.forename: B
authors.initials: B
authors.lastname: Pereira
authors.affiliation: Department of Surgery, Division of Trauma and Critical Care, Cedars-Sinai Medical Center, Los Angeles 90048, USA.
authors.forename: A
authors.initials: A
authors.lastname: Lamb
authors.affiliation: Department of Neurology, Cedars-Sinai Medical Center, Los Angeles 90048, USA.
authors.forename: L
authors.initials: L
authors.lastname: Zhao
authors.affiliation: Department of Neurology, Cedars-Sinai Medical Center, Los Angeles 90048, USA.
authors.forename: I-F
authors.initials: IF
authors.lastname: Lei
authors.affiliation: Department of Neurology, Cedars-Sinai Medical Center, Los Angeles 90048, USA.
authors.forename: J
authors.initials: J
authors.lastname: Bai
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D002545:Brain Ischemia / Q000209:etiology / Q000378:metabolism* / Q000517:prevention & control; D002421:Caudate Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D004195:Disease Models, Animal; D020244:Infarction, Middle Cerebral Artery / Q000150:complications; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D009474:Neurons / Q000378:metabolism*; D000066829:Neuroprotection; D051381:Rats; D017207:Rats, Sprague-Dawley; D055647:Rats, Transgenic; D044463:Receptor, PAR-1 / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism*
chemical_list: D044463:Receptor, PAR-1; D013917:Thrombin
keywords: cerebral ischemia; lentivirus knock-down; oxygen–glucose deprivation; protease activated receptor-1; thrombin