Improved Reperfusion and Vasculoprotection by the Poly(ADP-Ribose)Polymerase Inhibitor PJ34 After Stroke and Thrombolysis in Mice.

Benefits from thrombolysis with recombinant tissue plasminogen activator (rt-PA) after ischemic stroke remain limited due to a narrow therapeutic window, low reperfusion rates, and increased risk of hemorrhagic transformations (HT). Experimental data showed that rt-PA enhances the post-ischemic activation of poly(ADP-ribose)polymerase (PARP) which in turn contributes to blood-brain barrier injury. The aim of the present study was to evaluate whether PJ34, a potent PARP inhibitor, improves poor reperfusion induced by delayed rt-PA administration, exerts vasculoprotective effects, and finally increases the therapeutic window of rt-PA. Stroke was induced by thrombin injection (0.75 UI in 1 µl) in the left middle cerebral artery (MCA) of male Swiss mice. Administration of rt-PA (0.9 mg kg-1) or saline was delayed for 4 h after ischemia onset. Saline or PJ34 (3 mg kg-1) was given intraperitoneally twice, just after thrombin injection and 3 h later, or once, 3 h after ischemia onset. Reperfusion was evaluated by laser Doppler, vascular inflammation by immunohistochemistry of vascular cell adhesion molecule-1 (VCAM-1) expression, and vasospasm by morphometric measurement of the MCA. Edema, cortical lesion, and sensorimotor deficit were evaluated. Treatment with PJ34 improved rt-PA-induced reperfusion and promoted vascular protection including reduction in vascular inflammation (decrease in VCAM-1 expression), HT, and MCA vasospasm. Additionally, the combined treatment significantly reduced brain edema, cortical lesion, and sensorimotor deficit. In conclusion, the combination of the PARP inhibitor PJ34 with rt-PA after cerebral ischemia may be of particular interest in order to improve thrombolysis with an extended therapeutic window.

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

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.

Prevention of rt-PA induced blood-brain barrier component degradation by the poly(ADP-ribose)polymerase inhibitor PJ34 after ischemic stroke in mice.

Recombinant tissue plasminogen activator (rt-PA) is the only pharmacological treatment approved for thrombolysis in patients suffering from ischemic stroke, but its administration aggravates the risk of hemorrhagic transformations. Experimental data demonstrated that rt-PA increases the activity of poly(ADP-ribose)polymerase (PARP). The aim of the present study was to investigate whether PJ34, a potent (PARP) inhibitor, protects the blood-brain barrier components from rt-PA toxicity. In our mouse model of cerebral ischemia, administration of rt-PA (10 mg/kg, i.v.) 6h after ischemia aggravated the post-ischemic degradation of ZO-1, claudin-5 and VE-cadherin, increased the hemorrhagic transformations (assessed by brain hemoglobin content and magnetic resonance imaging). Furthermore, rt-PA also aggravated ischemia-induced functional deficits. Combining PJ34 with rt-PA preserved the expression of ZO-1, claudin-5 and VE-cadherin, reduced the hemorrhagic transformations and improved the sensorimotor performances. In vitro studies also demonstrated that PJ34 crosses the blood-brain barrier and may thus exert its protective effect by acting on endothelial and/or parenchymal cells. Thus, co-treatment with a PARP inhibitor seems to be a promising strategy to reduce rt-PA-induced vascular toxicity after stroke.