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

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 main 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 extravasated 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). Laser Doppler flowmetry (Perimed, anti–zonula occludens (ZO-1; Invitrogen, Camarillo, CA), Stockholm, Sweden) was used in all mice to confirm induction

190 Volume 73, No. 2 Wang et al: rADAMTS13 Reduces tPA-Induced Hemorrhage of ischemia and reperfusion. To increase the extent of cerebral Western Blotting hemorrhage, D-glucose (6ml/kg at 50% wt/vol) was injected in- Ischemic hemispheric brain tissues and matching tissue from traperitoneally 15 minutes before MCAO. Glucose was used sham-operated mice were dissected. The brain tissues were ho- because animal and human studies have shown that hyperglyce- mogenized in RIPA lysis buffer (Millipore) including protease in- mia increases the risk of tPA-associated cerebral hemorrhage af- hibitor cocktails (Roche Diagnostics, Mannheim, Germany). Af- ter ischemic stroke.26,28,29 After 2 hours of MCAO, tPA ter centrifugation, protein concentration was determined with (10mg/kg) or PBS was administered as an intravenous bolus the BCA protein assay (Thermo Scientific). Nuclear proteins injection of 1mg/kg followed by a 9mg/kg infusion for 30 were isolated using the NE-PER nuclear extraction kit (Pierce minutes with a syringe infusion pump (World Precision Instru- Biotechnology, Rockford, IL). Equal amounts of protein samples ments). VWF (3ll of 540ng) or PBS was injected intraventricu- were loaded on 8%, 10%, and 12% Tris-glycine gel, electropho- larly 90 minutes after MCAO. rADAMTS13 (100ng in 3ll resed, and transferred onto polyvinylidene difluoride membranes. PBS) or PBS was injected 3 hours after MCAO into the right Membranes were blocked with 5% nonfat dry milk in Tris-buf- lateral ventricle. Wortmannin (15lg/kg in 100ll saline contain- fered saline with 0.1% Tween 20 and probed with primary anti- ing 1% dimethyl sulfoxide [DMSO]) or vehicle (saline contain- bodies, followed by incubation with horseradish peroxidase–con- ing 1% DMSO) was administered intravenously immediately jugated secondary antibodies. Signals were detected with an before MCAO. Fasudil (10mg/kg in 100ll saline) or saline was enhanced chemiluminescence solution (Millipore) and quantified administered intravenously 30 minutes after MCAO. At 23 by scanning densitometry using a Bio-Image Analysis System hours after MCAO, mice were injected intravenously with 2ml/ (Bio-Rad, Hercules, CA). For the loading control, the same kg body weight of 2% Evans blue dye, and the amount of membrane was blotted with anti–b-actin antibody. extravasated Evans blue in the ipsilateral ischemic hemispheres was evaluated as above. Zymography Analyses Equal amounts of protein samples (30lg) were mixed with so- Behavioral Measurements dium dodecyl sulfate sample buffer (Invitrogen) and loaded At 24 hours after MCAO, mice were examined and scored onto 10% Tris-glycine gels (Invitrogen) containing 0.1% gela- blindly for neurological deficit as follows: 0, no neurological defi- tin. Human MMP-9 standard was also loaded in each gel. After cit; 1, failure to fully extend left forepaw; 2, reduced resistance to electrophoresis, gels were washed with renaturing buffer (Invi- lateral push; 3, spontaneous circling to left; 4, absence of sponta- trogen) for 1 hour and incubated in developing buffer (Invitro- neous movement or unconsciousness. In the locomotor activity gen) at 37C for 48 hours. Gels were stained with 0.5% Coo- test, mice were individually placed in a 27.3 27.3 40cm test massie blue R-250 for 1 hour and destained appropriately. environment for 30 minutes (Med Associates, St Albans, VT). MMP-9 band intensity was quantified and normalized to The test environment contained three 16-beam infrared arrays human MMP-9 standard. spaced 2.9cm apart. The animals’ movements were recorded by a digital video and analyzed via SOF-811 analyzer software (Med Immunostaining Associates). Locomotor activity was calculated using the total Twenty-four hours after MCAO, mice were killed by overdose number of infrared beams crossed by the mice. of chloral hydrate, and perfused transcardially with ice-cold PBS and 4% paraformaldehyde. The brains were removed and Quantification of Cerebral Hemorrhage cryoprotected in 30% sucrose in PBS. Brain cryosections After measurement of the other variables at 24 hours after (20lm) were blocked with 1% bovine serum albumin and 5% MCAO, mice were killed by overdose of chloral hydrate and normal donkey serum in PBS and were incubated with a com- perfused transcardially with ice-cold PBS. Drabkin reagent bination of antibodies of rabbit anti-VEGF and rat anti-CD31 (500ll) was added to the ischemic hemispheric brain tissue of or rabbit anti-RhoA and mouse anti-GFAP overnight at 4C. each mouse, followed by homogenization and centrifugation at The sections were then rinsed and incubated with a mixture of 13,000rpm for 30 minutes. Optical density of supernatants was Alexa Fluor 594 donkey antirabbit IgG and Alexa Fluor 488 measured by spectrophotometry (Thermo BioMate 3S; Thermo donkey antirat IgG or Alexa Fluor 488 donkey antimouse IgG Scientific) at 540nm. Hemorrhage volume was expressed in for 1 hour at room temperature. Images were obtained using an equivalent units by comparison with a reference curve generated Olympus (Tokyo, Japan) FV1000 confocal microscope and an using homologous blood as previously described.24,30 Olympus BX 51 microscope. For the quantification of VEGF- positive vessels, 5 nonoverlapping images in the peri-infarct Measurements of VWF Levels in Plasma area were digitized using a 40 objective. The images were Blood was collected 6 hours after MCAO by retro-orbital processed by NIH ImageJ and contrast-enhanced to clearly dif- bleeding into polypropylene tubes containing 3.8 % trisodium ferentiate positivity from background. The thresholded CD31 citrate. Platelet-poor plasma was prepared by centrifugation. and VEGF images were superimposed in 2 different layers. The VWF was measured using an enzyme-linked immunoassay CD31-positive area was selected, and the mean value of the (ELISA) kit (Ramco Laboratories, Stafford, TX) according to pixels of the VEGF image was calculated within the CD31 the manufacturer’s instructions, and expressed as a percentage selection.32 Data are presented as a percentage of the area of relative to a present plasma calibration standard.31 double positive VEGF and CD31 cells in reference to the total

February 2013 191 ANNALS of Neurology

also induces BBB permeability under nonischemic conditions, we quantified the extravasation of Evans blue dye in mice 1 hour after intraventricular injection of VWF. This analysis showed that intraventricular injection of VWF dose-dependently increased BBB permeability, with a significant effect at 540ng. Moreover, the VWF-cleaving protease rADAMTS13 completely blocked the VWF (540ng)-induced increase in BBB permeability. We therefore used 540ng of VWF in the following experiments. Coinjection of tPA together with VWF was not different with either injection alone, suggesting that the 2 agents may act within a common pathway. Next, we determined the effect of rADAMTS13 on the tPA-induced increases in BBB permeability. As expected, intraventricular coinjection of rADAMTS13 at 50 and 100ng significantly reduced the extravasation of Evans blue dye in tPA-treated mice. Because 100ng was more effective, we used 100ng of rADAMTS13 in the remaining studies. Injection with rADAMTS13 alone did not affect BBB permeability.

FIGURE 1: Recombinant ADAMTS13 (rADAMTS13) blocked rADAMTS13 Blocked tPA-Induced BBB tissue plasminogen activator (tPA)-induced blood–brain barrier permeability in the absence of ischemia. (A) Quantita- Disruption after Cerebral Ischemia tion of Evans blue dye 1 hour after intraventricular injection To investigate the role of rADAMTS13 on tPA-mediated of either phosphate-buffered saline (PBS), tPA, von Wille- degradation of BBB components after stroke, we sub- brand factor (VWF), or tPA in combination with VWF. (B) jected mice to 45 minutes of focal cerebral ischemia Quantitation of Evans blue dye 1 hour after intraventricular injection of different doses of VWF. (C) Quantitation of using the MCAO model, and examined the expression of Evans blue dye 1 hour after intraventricular injection of ei- ZO-1, a major tight junction protein, and collagen IV, a ther PBS, VWF, or VWF in combination with 100 ng major vascular basal membrane protein. We found that rADAMTS13 (rATS). (D) Quantitation of Evans blue dye 1 hour after intraventricular injection of either PBS, tPA, tPA cerebral ischemia significantly reduced ZO-1 and moder- in combination with 50 or 100 ng rADAMTS13, or 100 ng ately reduced collagen IV (Fig 2). Intravenous injection rADAMTS13. Values are mean 6 standard deviation. n 5 7– of tPA 2 hours after MCAO resulted in a substantial 8 per group; *p < 0.05. reduction of both ZO-1 and collagen IV compared with PBS-treated mice. However, intraventricular injection of area of CD31 positivity. The number of RhoA-GFAP double- labeled cells in the peri-infarct area was counted using a 40 rADAMTS13 3 hours after MCAO preserved the loss of objective. For each brain, 3 sections (2mm apart) from the is- ZO-1 and collagen IV caused by tPA. We further chemic hemisphere were analyzed. observed that treatment with tPA significantly increased the extravasation of Evans blue dye in the ischemic hemi- Statistical Analysis sphere compared with vehicle injection. Injection of Values are represented as mean 6 standard deviation and were rADAMTS13 reversed the alteration of BBB permeability analyzed using 1-way analysis of variance followed by the Bon- in mice treated with tPA. rADAMTS13 alone did not ferroni multiple comparison test. The differences in behavior affect the BBB permeability after cerebral ischemia. were assayed using the Kruskal–Wallis test followed by the Dunn multiple comparison test. When 2 groups were com- Effects of rADAMTS13 on Cerebral Hemorrhage pared, the unpaired 2-tailed Student t test was used. A proba- and Neurological Function after Cerebral bility of p < 0.05 was considered statistically significant. Ischemia We asked whether rADAMTS13 could reduce the extent Results of tPA-associated cerebral hemorrhage. By using a spec- rADAMTS13 Blocked tPA-Induced Increases in trophotometric hemoglobin assay, we found that infusion BBB Permeability in the Absence of Ischemia of tPA 2 hours after MCAO resulted in a significant We first confirmed that intraventricular injection of tPA increase in hemorrhage 24 hours after stroke (Fig 3). increased BBB permeability in the absence of cerebral is- Intraventricular injection of rADAMTS13 3 hours after chemia (Fig 1), as reported.26,33 To test whether VWF MCAO blocked tPA-induced hemorrhage. Treatment

192 Volume 73, No. 2 Wang et al: rADAMTS13 Reduces tPA-Induced Hemorrhage

In addition, mice treated with tPA showed more severe neurological deficits as assayed by the neurological scores and motor function than the PBS-treated mice. However, the neurological scores and motor function were significantly better in the rADAMTS13 þ tPA group than in the tPA group (Fig 4).

rADAMTS13 Downregulated tPA-Evoked VEGF Induction in Vascular Endothelium MMP-9 promotes injury of the BBB and hemorrhage after stroke.12 To examine whether rADAMTS13

FIGURE 2: Recombinant ADAMTS13 (rADAMTS13) blocked tissue plasminogen activator (tPA)-induced blood–brain barrier disruption after cerebral ischemia. (A, C) Representative immunoblots of tight junction protein zonula occludens (ZO- 1) and basal membrane protein collagen IV from sham-operated mice, and mice treated with vehicle, tPA, and tPA 1 rADAMTS13 (rATS) 24 hours after stroke. (B, D) Quantita- tive determinations of ZO-1 and collagen IV for each group. Values are mean 6 standard deviation (SD). n 5 4 for sham- operated group, n 5 6 for other groups; *p < 0.05. (E) Representative images of Evans blue extravasation 24 hours after stroke in mice treated with vehicle, tPA, tPA 1 rADAMTS13, and rADAMTS13. (F) Quantification of Evans blue fluorescent intensity for each group. Values are mean 6 SD. n 5 7 per group; *p < 0.05. MCAO 5 middle cerebral artery occlusion; PBS 5 phosphate-buffered saline.

FIGURE 3: Effects of recombinant ADAMTS13 (rADAMTS13) with rADAMTS13 alone did not significantly affect the on tPA-induced cerebral hemorrhage after cerebral ischemia. hemorrhage volume. Next, we investigated the effect of (A) Representative images of the dorsal surface and a coro- rADAMTS13 on tPA-induced hemorrhage through its nal section show cerebral hemorrhage 24 hours after stroke in mice treated with vehicle, tissue plasminogen activator action on VWF. ELISA analysis indicated that treatment (tPA), tPA 1 rADAMTS13 (rATS), and rADAMTS13. Note the with tPA at 2 hours significantly increased plasma levels massive hemorrhagic transformation (arrows) within the is- of VWF compared with PBS-treated mice 6 hours after chemic area in mice treated with tPA. (B) Quantification of cerebral hemorrhage by spectrophotometric hemoglobin MCAO. We then observed that intraventricular injection assay. Values are mean 6 standard deviation (SD). n 5 8 per of VWF resulted in a 2.3-fold increase in hemorrhage group; *p < 0.05. (C) Plasma levels of von Willebrand factor volume compared with PBS-treated mice. Treatment (VWF) 6 hours after stroke in mice treated with vehicle and 6 5 < with VWF and tPA had no additive effects, indicating tPA. Values are mean SD. n 5–7 per group; *p 0.05. (D) Representative images of the dorsal surface show cere- that the tPA-induced hemorrhage requires VWF. More- bral hemorrhage 24 hours after stroke in mice treated with over, injection of VWF reversed the antihemorrhagic vehicle, VWF, VWF 1 tPA, and VWF 1 tPA 1 rADAMTS13. effect of rADAMTS13 on tPA-induced bleeding. (E) Quantification of cerebral hemorrhage by spectrophotometric hemoglobin assay. Values are mean 6 SD. n 5 6-7 Together, these data demonstrated that rADAMTS13 per group; *p < 0.05. MCAO 5 middle cerebral artery occlu- exerts its effect on tPA-induced hemorrhage via VWF. sion; PBS 5 phosphate-buffered saline.

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endothelium in the peri-infarct area was reduced by rADAMTS13 (see Fig 6C, D). Because both Ang-1 and Ang-2 were previously reported to regulate cerebrovascular permeability after cerebral ischemia,37 we assessed the levels of Ang-1 and Ang-2 in brain samples. Our results indicated that the levels of Ang-1 and Ang-2 (see Fig 6E) were comparable among groups, suggesting that the tPA-induced hemorrhage is not mediated by Ang-1 and Ang-2. FIGURE 4: Effects of recombinant ADAMTS13 (rADAMTS13) on tissue plasminogen activator (tPA)-induced neurological dysfunction after cerebral ischemia. (A) Neurological severity Inhibition of tPA-Induced VEGF Expression by score and (B) locomotor activity 24 hours after stroke in mice rADAMTS13 Was Associated with Akt and treated with vehicle, tPA, tPA 1 rADAMTS13 (rATS), and RhoA Pathways rADAMTS13. Values are mean 6 standard deviation. n 5 8 38,39 per group; *p < 0.05. MCAO 5 middle cerebral artery VEGF is regulated by Akt and RhoA pathways, both 13,40 occlusion. of which mediate ischemia-evoked BBB dysfunction. To test whether these factors have a role in tPA-mediated VEGF upregulation, we administered intravenously the attenuated tPA-induced hemorrhage through MMP-9, Akt inhibitor wortmannin and the RhoA inhibitor we performed gelatin zymographic assays in brain fasudil in mice subjected to cerebral ischemia and tPA extracts. We found that combination of rADAMTS13 treatment. We found that both wortmannin and fasudil and tPA significantly reduced MMP-9 activity compared suppressed tPA-induced VEGF upregulation (Fig 7). We with the tPA alone group (Fig 5A, B). NF-jB was then asked whether rADAMTS13 could inhibit tPA- reported to trigger upregulation of MMP-9 expression.11 induced phosphorylation of Akt and activation of RhoA. We measured nuclear translocation of NF-jB in ischemic Our Western blot analysis showed that cerebral ischemia brain at 24 hours after MCAO. Our results demon- induced Akt phosphorylation and increased the activity strated that combination treatment with rADAMTS13 and tPA did not significantly affect the nuclear NF-jB levels compared with tPA-treated mice (see Fig 5C, D). VEGF, the major proangiogenic factor, increased microvascular permeability, leading to exacerbated edema and hemorrhage after cerebral ischemia.34,35 We therefore examined whether tPA treatment could substantially affect VEGF expression. Western blot analysis revealed that cerebral ischemia significantly increased VEGF expression compared with the sham-operated group, which is consistent with previous reports.36 Mice treated with tPA showed an even greater increase of VEGF expression compared with PBS-treated mice (Fig 6). Double immunostaining revealed that VEGF was mainly colocalized with the CD31-positive blood vessels at 24 hours after stroke. As reported,36 VEGF immunoreactivity was also observed in astrocytes and neurons (data not FIGURE 5: Effects of recombinant ADAMTS13 (rADAMTS13) shown). We found that treatment with tPA significantly and tissue plasminogen activator (tPA) on matrix metalloproteinase-9 (MMP-9) and nuclear factor kappa B (NF-jB) expression increased the VEGF-immunostained vessels in the peri- after cerebral ischemia. (A) Representative zymographic analy- infarct area compared with PBS-treated mice. sis of brain extracts from sham-operated mice, and mice treated Given the evidence that rADAMTS13 blocked tPA- with vehicle, tPA, and tPA 1 rADAMTS13 (rATS) 24 hours after induced cerebral hemorrhage, we examined whether stroke. (B) Quantitative determination of zymographic gels for each group. Values are mean 6 standard deviation (SD). n 5 5 rADAMTS13 could decrease tPA-mediated upregulation per group; *p < 0.05. (C) Representative immunoblots of NF-jB of VEGF. Western blot analysis showed that treatment expression from sham-operated mice, and mice treated with ve- with rADAMTS13 suppressed the increase in VEGF hicle, tPA, and tPA 1 rADAMTS13 24 hours after stroke. (D) Quantitative determination of nuclear NF-jB expression for activation by tPA (see Fig 6A, B). Consistent with this, each group. Values are mean 6 SD. n 5 5pergroup;*p < 0.05. tPA-induced VEGF immunoreactivity in vascular MCAO 5 middle cerebral artery occlusion.

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Immunohistochemical analysis showed that RhoA was localized with astrocytes (see Fig 7I, arrows) and neuronlike cells (see Fig 7I, arrowheads) in the peri-infarct area after stroke. Because astrocytes play an important part in the formation and maintenance of the BBB,13 we counted the number of RhoA-GFAP double-labeled cells in the peri-infarct area at 24 hours after MCAO. Com- pared with tPA-treated mice, we observed a significant decrease in the number of RhoA immunoreactive astrocytes in mice treated with rADAMTS13 and tPA (see Fig 7I, J).

Discussion Cerebral hemorrhage associated thrombolytic therapy in acute stroke continues to present a major clinic prob- lem.2–4 Although the mechanisms underlying tPA- induced hemorrhage are still unclear, it has been sug- gested that it occurs as a result of BBB disruption.2,14,15 In the present study, we showed that VWF directly induced opening of the BBB under nonischemic conditions. Specifically, we demonstrated that the VWF-cleaving protease rADAMTS13 blocked tPA-potentiated deterioration of cerebrovascular integrity, thereby reducing tPA-mediated hemorrhage after ischemic stroke. In the CNS, both tPA and VWF are expressed abundantly in endothelial cells.4,20 Although the association of tPA with BBB permeability is now well estab- lished, the specific roles of VWF on the BBB remain FIGURE 6: Recombinant ADAMTS13 (rADAMTS13) downre- unclear. Here, we demonstrated that direct injection of gulated tissue plasminogen activator (tPA)-evoked vascular endothelial growth factor (VEGF) induction in vascular endo- VWF into the lateral ventricle of nonischemic mice thelium. (A) Representative immunoblots of VEGF expres- increased BBB permeability to a similar extent as tPA. sion from sham-operated mice, and mice treated with This result suggests that VWF may be a natural regulator vehicle, tPA, and tPA 1 rADAMTS13 (rATS) 24 hours after stroke. (B) Quantitative determination of VEGF expression of the cerebrovascular permeability, as is tPA. Previous for each group. Values are mean 6 standard deviation (SD). studies have reported that VWF protein was upregulated n 5 4 for sham-operated group, n 5 6 for other groups; *p in patients with ischemic stroke.21 Furthermore, in < 0.05. (C) Double immunostaining for VEGF and endothelial cells (CD31) 24 hours after stroke in mice treated with rodents, genetic deficiency of VWF or treatment with its vehicle, tPA, and tPA 1 rADAMTS13. Scale bar 5 12.5lm. cleaving protease rADAMTS13 decreased the extent of (D) Quantitative determination of the VEGF-immunostained ischemic brain injury.24,25,41 Given the evidence that vessels for each group. Values are mean 6 SD. n 5 5 per VWF is a key factor in thrombus formation after group; *p < 0.05. (E) Representative immunoblots of angio- 42,43 poietin (Ang)-1 and Ang-2 expression from sham-operated stroke, interaction of tPA with VWF seems to be mice, and mice treated with vehicle, tPA, and tPA 1 crucial during thrombolysis. In fact, recent reports have rADAMTS13 24 hours after stroke. MCAO 5 middle cere- revealed that thrombolytic therapy with tPA was accom- bral artery occlusion; PBS 5 phosphate-buffered saline. panied by elevated levels of VWF.23 In the present study, we observed that intraventricular injection of VWF in is- of RhoA. These effects were significantly amplified when chemic mice caused a significant increase in cerebral tPA was applied. In contrast, rADAMTS13 blocked tPA- hemorrhage. We then found that tPA treatment increased mediated phosphorylation of Akt and activation of the plasma levels of VWF after cerebral ischemia. There- RhoA. No differences of total Akt were detected among fore, we hypothesize that this elevated VWF could lead groups. These data suggest that rADAMTS13 may to deterioration of cerebrovascular integrity after stroke. inhibit the tPA–VEGF pathway by suppressing the phos- Consistent with this, we showed that ischemic degrada- phorylation of Akt and activation of RhoA. tion of critical components of the BBB were exacerbated

February 2013 195 ANNALS of Neurology by tPA treatment, and these effects were reversed by ever, because hyperglycemia was induced to increase cere- rADAMTS13. Correspondingly, tPA-induced BBB dis- bral hemorrhage in the current study, caution should be ruption and cerebral hemorrhage were inhibited by taken in extrapolating the results to stroke patients in rADAMTS13. Because thrombolysis with tPA leads to general. some degree of endothelial cell damage,44 the effect of VEGF mediates tPA-associated activation of MMP- rADAMTS13 may result from the cleavage of ultralarge 9 and degradation of the BBB components,35,36 and VWF secreted locally from activated endothelium. How- pharmacologic inhibition of VEGF reduced tPA-associated hemorrhage.36 Recent data also showed that rADAMTS13 could inhibit angiogenesis through its interaction with VEGF.45 In the present study, we observed that tPA significantly amplified VEGF expression in vascular endothelium in the peri-infarct area after cerebral ischemia. tPA selectively induced cerebral hemorrhage in the ischemic area, including the peri-infarct area.46 This result may suggest that newly synthesized VEGF in endothelial cells in the peri-infarct area after tPA administration resulted in the disruption of brain vessels. We also demonstrated that treatment with rADAMTS13 reversed the upregulation of VEGF and MMP-9 caused by tPA. Taken together, these findings suggest that rADAMTS13 may prevent proteolysis of critical BBB components via inhibition of the VEGF– MMP-9 pathway. However, because VEGF also partici- pates in tissue repair by promoting angiogenesis in the later phase of stroke,34 a caveat may arise regarding the potential detrimental effects of prolonged VEGF inhibition. Further study would be important to define the time and duration of optimal inhibition of VEGF to avoid compromising the later neurovascular remodeling. The serine/threonine protein kinase Akt was shown to play a role not only in neuronal survival but also in

FIGURE 7: Inhibition of tissue plasminogen activator (tPA)- induced vascular endothelial growth factor (VEGF) expression by recombinant ADAMTS13 (rADAMTS13) was associated with Akt and RhoA pathways. (A, C) Representative immunoblots of VEGF expression 24 hours after stroke in the ischemic brain of mice treated with tPA and tPA 1 wortmannin or fasudil. (B, D) Quantitative determination of VEGF for each group. Values are mean 6 standard deviation (SD). n 5 5 per group; *p < 0.05. (E, G) Representative immunoblots of phosphorylated Akt (pAkt), total Akt, and RhoA from sham-operated mice, and mice treated with vehicle, tPA, and tPA 1 rADAMTS13 (rATS) 24 hours after stroke. (F, H) Quantitative determination of phosphorylated Akt and RhoA for each group. Values are mean 6 SD. n 5 4 for sham-operated group, n 5 6 for other groups; *p < 0.05. (I) Double immunostaining for RhoA (red) and astrocytes (glial fibrillary acidic protein [GFAP], green) after stroke in mice treated with vehicle, tPA, and tPA 1 rADAMTS13. The arrows indicate RhoA–GFAP double-labeled cells, and the arrowheads indicate neuronlike cells. Scale bar 5 15lm. (J) Quantitative determination of the number of RhoA–GFAP double-labeled cells for each group. Values are mean 6 SD. n 5 5 per group; *p < 0.05. MCAO 5 middle cerebral artery occlusion; PBS 5 phosphate-buffered saline.

196 Volume 73, No. 2 Wang et al: rADAMTS13 Reduces tPA-Induced Hemorrhage regulation of the BBB permeability.13,38 Furthermore, it stroke: where do we go from here? A cumulative meta-analysis. Stroke 2003;34:1437–1442. was already demonstrated that the Akt pathway was 4. Vivien D, Buisson A. Serine protease inhibitors: novel therapeutic involved in VEGF-induced BBB permeability after targets for stroke? J Cereb Blood Flow Metab 2000;20:755–764. 38 stroke. The Rho–associated protein kinase pathway plays 5. Lo EH, Dalkara T, Moskowitz MA. Mechanisms, challenges and 47 important roles in VEGF-induced angiogenesis. The opportunities in stroke. Nat Rev Neurosci 2003;4:399–415. small guanosine triphosphatase RhoA was upregulated af- 6. Nicole O, Docagne F, Ali C, et al. The proteolytic activity of tis- ter ischemia and hemorrhage,40,48 and inactivation of sue-plasminogen activator enhances NMDA receptor-mediated signaling. Nat Med 2001;7:59–64. 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