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Tissue-Type Plasminogen Activator Crosses the Intact Blood-Brain Barrier by Low-Density Lipoprotein Receptor– Related Protein-Mediated Tissue-Type Plasminogen Activator Crosses the Intact Blood-Brain Barrier by Low-Density Lipoprotein Receptor– Related Protein-Mediated Transcytosis by Karim Benchenane, Vincent Berezowski, Carine Ali, Mónica Fernández-Monreal, José P. López-Atalaya, Julien Brillault, Julien Chuquet, André Nouvelot, Eric T. Mac. Kenzie, Guojun Bu, Roméo Cecchelli, Omar Touzani, and Denis Vivien Circulation Volume 111(17): 2241 -2249 May 3, 2005 Copyright © American Heart Association, Inc. All rights reserved.

Figure 1. Intravenous (iv) or intrastriatal (is) injection of t. PA potentiates NMDA-induced lesion. Figure 1. Intravenous (iv) or intrastriatal (is) injection of t. PA potentiates NMDA-induced lesion. a, Rats were injected stereotaxically, into left striatum, with either NMDA alone (50 nmol), t. PA alone (3 μg), or NMDA plus t. PA (n=5). b, Same protocol as described above, together with intravenous injection of t. PA (1 mg/kg or 10 mg/kg) or vehicle (n=10, 7, and 10). c, Same protocol with either NMDA alone or with PAI-1 together with intravenous injection of t. PA (1 mg/kg) or vehicle (n=7 to 10 in each group). *P<0. 05, **P<0. 01, ***P<0. 001, ANOVA with Bonferroni correction. Karim Benchenane et al. Circulation. 2005; 111: 2241 -2249 Copyright © American Heart Association, Inc. All rights reserved.

Figure 2. Intrastriatal injection of NMDA with or without intravenous injection of t. PA Figure 2. Intrastriatal injection of NMDA with or without intravenous injection of t. PA fails to induce BBB breakdown after 2 hours. 3 D reconstruction with confocal microscopy (× 20) after (a) intravenous injection of FITC-dextran (green; 77 k. Da), (b) intravenous injection of t. PA (1 mg/kg) together with FITC-dextran, (c) intrastriatal injection of NMDA 30 minutes before FITC-dextran injection, and (d) intrastriatal injection of NMDA 30 minutes before intravenous injection of both t. PA and FITC-dextran. e, Image showing extravasation of FITC-dextran at site of burr hole as positive control. Karim Benchenane et al. Circulation. 2005; 111: 2241 -2249 Copyright © American Heart Association, Inc. All rights reserved.

Figure 3. Blood-derived t. PA reaches brain parenchyma after intravenous injection. a, Biotinylated t. Figure 3. Blood-derived t. PA reaches brain parenchyma after intravenous injection. a, Biotinylated t. PA (biot-t. PA) was injected intravenously (iv) after intrastriatal injection of NMDA or no injection, as described in Methods. Karim Benchenane et al. Circulation. 2005; 111: 2241 -2249 Copyright © American Heart Association, Inc. All rights reserved.

Figure 4. Blood-derived t. PA crosses intact BBB. Biotinylated t. PA (i through n) Figure 4. Blood-derived t. PA crosses intact BBB. Biotinylated t. PA (i through n) or biotinylated albumin (a through h) was injected intravenously in control condition (a through d and i through n) or in mechanically induced BBB disruption (e through h). Karim Benchenane et al. Circulation. 2005; 111: 2241 -2249 Copyright © American Heart Association, Inc. All rights reserved.

Figure 5. t. PA crosses BBB in vitro. a, Schematic representation of in vitro Figure 5. t. PA crosses BBB in vitro. a, Schematic representation of in vitro model of BBB. b, t. PA was applied in luminal compartment. t. PA activity in abluminal media was analyzed by caseinplasminogen zymography assay at different times. Karim Benchenane et al. Circulation. 2005; 111: 2241 -2249 Copyright © American Heart Association, Inc. All rights reserved.

Figure 6. t. PA has no effect on integrity of BBB in vitro. a, Figure 6. t. PA has no effect on integrity of BBB in vitro. a, Immunostaining of endothelial cells with antibody raised against ZO-1, a protein of tight junctions, treated or not treated with t. PA (4 hours, 20 μg/m. L). Karim Benchenane et al. Circulation. 2005; 111: 2241 -2249 Copyright © American Heart Association, Inc. All rights reserved.

Figure 7. t. PA crosses BBB in vitro independently of its proteolytic activity. a, Figure 7. t. PA crosses BBB in vitro independently of its proteolytic activity. a, Biotinylated t. PA (biot-t. PA) or biotinylated t. PA plus t. PA Stop was applied in luminal compartment. t. PA activity in luminal compartment was analyzed by fluorogenic substrate assay. Karim Benchenane et al. Circulation. 2005; 111: 2241 -2249 Copyright © American Heart Association, Inc. All rights reserved.

Figure 8. t. PA crosses BBB in vitro by active mechanism. t. PA was Figure 8. t. PA crosses BBB in vitro by active mechanism. t. PA was applied in luminal compartment at 37°C or 4°C. Karim Benchenane et al. Circulation. 2005; 111: 2241 -2249 Copyright © American Heart Association, Inc. All rights reserved.

Figure 9. t. PA crosses BBB in vitro by receptor-dependent pathway. a, t. PA Figure 9. t. PA crosses BBB in vitro by receptor-dependent pathway. a, t. PA was applied in luminal compartment at different doses. Karim Benchenane et al. Circulation. 2005; 111: 2241 -2249 Copyright © American Heart Association, Inc. All rights reserved.

Figure 10. t. PA crosses BBB in vitro by LRP-mediated transcytosis. a, t. PA Figure 10. t. PA crosses BBB in vitro by LRP-mediated transcytosis. a, t. PA alone or in presence of mannose (100 mmol/L) or RAP (500 nmol/L) was applied in luminal compartment. t. PA activity in luminal compartment was analyzed by fluorogenic substrate assay. Karim Benchenane et al. Circulation. 2005; 111: 2241 -2249 Copyright © American Heart Association, Inc. All rights reserved.