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Recombinant ADAMTS13 Reduces Tissue Plasminogen Activator-Induced Hemorrhage After Stroke in Mice

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ORIGINAL ARTICLE Recombinant ADAMTS13 Reduces Tissue Plasminogen Activator-Induced Hemorrhage after Stroke in Mice Lixiang Wang, MD, Wenying Fan, MD, PhD, Ping Cai, MS, Mengchen Fan, BS, Ximin Zhu, BS, Yiqin Dai, BS, Chungang Sun, MD, Yannan Cheng, BS, Ping Zheng, MD, PhD, and Bing-Qiao Zhao, MD, PhD Objective: Tissue plasminogen activator (tPA) is approved for treatment of acute ischemic stroke, but it increases the risk of cerebral hemorrhage. Accumulating evidence suggests that von Willebrand factor (VWF) plays a pivotal role in thrombus formation and microcirculatory disturbances after ischemic stroke. By cleaving VWF, ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) protects mice from stroke. Therefore, we hypothesized that recombinant ADAMTS13 (rADAMTS13) could increase the safety of tPA thrombolysis in stroke. Methods: We examined blood–brain barrier (BBB) permeability after intraventricular injection of tPA, VWF, and rADAMTS13 in nonischemic mice. We investigated the role of rADAMTS13 on reducing tPA-induced BBB dysfunction and cerebral hemorrhage in a mouse stroke model. Results: Intraventricular injection of tPA or VWF under nonischemic conditions resulted in a significant increase in BBB permeability. In contrast, rADAMTS13 blocked both tPA- and VWF-induced BBB opening. BBB disruption following stroke was exacerbated by intravenous administration of tPA, but this was attenuated by injection of rADAMTS13. Correspondingly, tPA-associated hemorrhage after stroke was significantly reduced by rADAMTS13. The antihemorrhagic effect of rADAMTS13 was reversed by injection of recombinant VWF. We also showed that rADAMTS13 inhibited tPA-mediated upregulation of vascular endothelial growth factor (VEGF) in vascular endothelium after stroke. The upregulation of VEGF was suppressed by either an Akt inhibitor wortmannin or a Rho kinase inhibitor fasudil. Furthermore, rADAMTS13 downregulated tPA-induced phosphorylation of Akt and activation of RhoA. Interpretation: These findings demonstrate that the VWF-cleaving protease rADAMTS13 reduced tPA-induced hemorrhage by regulating BBB integrity, and suggest that this effect may occur through the Akt/RhoA-mediated VEGF pathways. ANN NEUROL 2013;73:189–198 troke is a leading cause of death and disability world- tPA is a serine protease that plays a critical role in Swide. Thrombolytic therapy with tissue plasminogen the homeostasis of blood coagulation and fibrinolysis.4 activator (tPA) is beneficial for acute ischemic stroke.1 Besides regulating normal central nervous system (CNS) However, the use of tPA is associated with increased risk physiology and neuronal plasticity,5 tPA also plays a role of cerebral hemorrhage, especially when tPA is adminis- in CNS pathology. tPA promoted excitotoxic neuronal tered beyond the first few hours after symptom onset.2–4 death6 and increased ischemic brain injury,7 and genetic Cerebral hemorrhage is the most feared complication of ablation of tPA or pharmacologic inhibition of tPA was tPA and an important obstacle of its use.3,5 Therefore, protected from cerebral ischemia.7,8 Furthermore, tPA any strategy for attenuating the severity of hemorrhage was shown to exacerbate ischemic endothelial injury and will have a significant impact on improving the outcomes blood–brain barrier (BBB) disruption by promoting ma- in patients with ischemic stroke. trix metalloproteinase-9 (MMP-9) and nuclear factor View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.23762 Received Feb 18, 2012, and in revised form Aug 13, 2012. Accepted for publication Sep 4, 2012. Address correspondence to Dr Zhao, State Key Laboratory of Medical Neurobiology, Fudan University, 138 Yixueyuan Road, Shanghai 200032, China. E-mail: [email protected] or Dr Fan, State Key Laboratory of Medical Neurobiology, Fudan University, 138 Yixueyuan Road, Shanghai 200032, China. E-mail: [email protected] From the State Key Laboratory of Medical Neurobiology, Shanghai Medical College and Institutes of Brain Science, Fudan University, Shanghai, China. VC 2012 American Neurological Association 189 ANNALS of Neurology kappa B (NF-jB) activation, Akt phosphorylation, and rabbit anti–collagen IV (Santa Cruz Biotechnology, Santa Cruz, extracellular matrix degradation.9–13 Experimental and CA), rabbit anti–vascular endothelial growth factor (VEGF; clinical evidence has indicated that disruption of the Abcam, Cambridge, MA), goat anti–angiopoietin (Ang)-1 BBB after tPA treatment was highly predictive for subse- (Santa Cruz Biotechnology), goat anti–Ang-2 (Santa Cruz Bio- quent cerebral hemorrhage.14,15 This suggests that inhibi- technology), rabbit anti–phospho-Akt (serine 473; Cell Signal- ing Technology, Beverly, MA), rabbit anti-Akt (Cell Signaling tion of tPA-mediated BBB disruption may represent a Technology), rabbit anti-RhoA (Santa Cruz Biotechnology), rat potential strategy to increase the safety of tPA thrombol- anti-CD31 (PECAM-1; BD Pharmingen, San Diego, CA), ysis for ischemic stroke. mouse anti–glial fibrillary acidic protein (GFAP; Millipore), von Willebrand factor (VWF) is a multimeric gly- rabbit anti–NF-jB (p65; Cell Signaling Technology), and rab- coprotein that is produced by endothelial cells and mega- bit anti–b-actin (Cell Signaling Technology). Secondary anti- 16 karyocytes. ADAMTS13 (a disintegrin and metallopro- bodies used were: Alexa Fluor 594 donkey antirabbit immuno- teinase with a thrombospondin type 1 motif, member globulin G (IgG), Alexa Fluor 488 donkey antirat IgG, and 13) is a zinc-containing metalloprotease present in Alexa Fluor 488 donkey antimouse IgG (Invitrogen). plasma.17,18 The only known substrate for ADAMTS13 is VWF. The most important function of this metallo- Animals protease is to cleave the newly secreted ultralarge forms Eight 10-week-old male C57BL/6J mice (Shanghai SLAC Lab- of VWF to the smaller, less adhesive multimeric forms, oratory Animal Co, Shanghai, China) weighing 23 to 26g were used in all experiments. All experimental procedures were thus downregulating the thrombotic process.19 In the approved by the Animal Care and Use Committee of Shanghai CNS, VWF is expressed abundantly in endothelial Medical College, Fudan University. cells20; however, little is known about the role of VWF in regulation of BBB permeability. Several studies have Intraventricular Injections and Cerebrovascular shown that plasma levels of VWF were elevated in dis- Permeability Evaluation eases associated with BBB disruption, such as ischemic Mice were anesthetized with 1 to 1.5% isoflurane in 30% oxy- stroke, intracerebral hemorrhage, and subarachnoidal gen, placed on a stereotactic frame (Narishige Scientific Instru- hemorrhage.21,22 Furthermore, elevated plasma levels of ment Laboratory, Tokyo, Japan). Intracerebroventricular injec- VWF were found after thrombolytic therapy in patients tions (3ll) of either phosphate-buffered saline (PBS), tPA with cardiovascular disease.23 Recently, we have shown (585ng), VWF (540ng), tPA in combination with VWF, tPA in that absence of VWF protected mice from cerebral ische- combination with rADAMTS13 (50 or 100ng), or mia.24 In contrast, ADAMTS13 deficiency resulted in rADAMTS13 (100ng) were given at the following coordinates from bregma: 0.2mm posterior, 1.0mm lateral, and 3.0mm larger infarcts,24,25 and treatment with recombinant ventral. The injections were performed over 15 minutes. The ADAMTS13 (rADAMTS13) reduced ischemic brain 26 24 dose of tPA was chosen based on previous studies. The effects injury. of rADAMTS13 on VWF-induced BBB permeability was In the present study, we showed that direct injec- analyzed in a different group of mice, which were injected tion of VWF into the cerebrospinal fluid of nonischemic intraventricularly with 3ll of rADAMTS13 (100ng) 10 minutes mice induced a rapid opening of the BBB. We also dem- after intraventricular injection of VWF (3ll of 540ng). Evans onstrated that the VWF-cleaving protease rADAMTS13 blue dye (4% in PBS, 4ml/kg) injections were given intrave- blocked tPA-induced loss of cerebrovascular integrity, nously and allowed to circulate for 60 minutes. Animals were and consequently decreased tPA-associated cerebral perfused transcardially with PBS, and their brains were removed hemorrhage. and placed in formamide for 72 hours. The amount of extrava- sated Evans blue dye was evaluated by spectrophotometry (Thermo BioMate 3S; Thermo Scientific, Waltham, MA) at Materials and Methods 620nm. Reagents and Antibodies Evans blue, Drabkin reagent, paraformaldehyde, D-glucose, Stroke Model formamide, and wortmannin were purchased from Sigma- Cerebral ischemia was induced as described previously.24,27 In Aldrich (St Louis, MO). Human recombinant tPA (Actilyse) brief, transient occlusion of the right middle cerebral artery was obtained from Boehringer Ingelheim (Mannheim, Ger- (MCAO) was induced with a 7.0 siliconized filament through many). Human VWF protein was from Haematologic Technol- the external carotid artery. After 45 minutes, the filament was ogies (Essex Junction, VT), and human rADAMTS13 was from removed to allow reperfusion. Mice were anesthetized with 1 to R&D Systems (Minneapolis, MN). Fasudil was from Calbio- 1.5% isoflurane in 30% oxygen. Body temperature was main- chem (San Diego, CA). Purified Human MMP-9 was from tained at 37 6 0.5C using a heating pad (World Precision Millipore (Billerica, MA). Primary antibodies used were rabbit Instruments, Sarasota, FL). 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