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Lack of TAFI increases brain damage and microparticle generation after thrombolytic therapy in ischemic stroke
TR-05990; No of Pages 6 Thrombosis Research xxx (2015) xxx–xxx
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Lack of TAFI increases brain damage and microparticle generation after thrombolytic therapy in ischemic stroke J. Orbe a,⁎, N. Alexandru b, C. Roncal a, M. Belzunce a, P. Bibiot a, J.A. Rodriguez a, J.C.M. Meijers c,d, A. Georgescu b, J.A. Paramo a a
Laboratory of Atherothrombosis, Program of Cardiovascular Diseases, CIMA-University of Navarra, Navarra Institute for Health Research (IdiSNA), Pamplona, Spain Pathophysiology and Pharmacology Department, Institute of Cellular Biology and Pathology ‘Nicolae Simionescu’ of Romanian Academy, Bucharest, Romania Experimental Vascular Medicine, Academic Medical Center, University of Amsterdam, the Netherlands d Department of Plasma Proteins, Sanquin Research, Amsterdam, the Netherlands b c
a r t i c l e
i n f o
Article history: Received 18 February 2015 Received in revised form 29 May 2015 Accepted 8 June 2015 Available online xxxx Keywords: TAFI Fibrinolysis Thrombolytic therapy Circulating cell-derived microparticles Inflammation
a b s t r a c t Background: Thrombin-activatable fibrinolysis inhibitor (TAFI) plays an important role in coagulation and fibrinolysis. Whereas TAFI deficiency may lead to a haemorrhagic tendency, data from TAFI knockout mice (TAFI −/−) are controversial and no differences have been reported in these animals after ischemic stroke. There are also no data regarding the role of circulating microparticles (MPs) in TAFI−/−. Objectives: to examine the effect of tPA on the rate of intracranial haemorrhage (ICH) and on MPs generated in a model of ischemic stroke in TAFI−/− mice. Methods: Thrombin was injected into the middle cerebral artery (MCA) to analyse the effect of tPA (10 mg/Kg) on the infarct size and haemorrhage in the absence of TAFI. Immunofluorescence for Fluoro-Jade C was performed on frozen brain slides to analyse neuronal degeneration after ischemia. MPs were isolated from mouse blood and their concentrations calculated by flow cytometry. Results: Compared with saline, tPA significantly increased the infarct size in TAFI−/− mice (p b 0.05). Although plasma fibrinolytic activity (fibrin plate assay) was higher in these animals, no macroscopic or microscopic ICH was detected. A positive signal for apoptosis and degenerating neurons was observed in the infarct area, being significantly higher in tPA treated TAFI−/− mice (p b 0.05). Interestingly, higher numbers of MPs were found in TAFI−/− plasma as compared to wild type, after stroke (p b 0.05). Conclusions: TAFI deficiency results in increased brain damage in a model of thrombolysis after ischemic stroke, which was not associated with bleeding but with neuronal degeneration and MP production. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma procarboxypeptidase that upon activation by thrombin or thrombin/ thrombomodulin turns into a potent antifibrinolytic enzyme [1–3]. The activation of TAFI by thrombin implies that the coagulation system plays a role in the regulation of fibrinolysis and that disturbances in TAFI levels will affect the rate of lysis [4–6]. Since clot stabilisation seems to be a requisite for the prevention of bleeding, it could be assumed that TAFI deficiency would lead to a hemorrhagic tendency by inducing premature fibrinolysis [7]. In vivo evidence for the role of TAFI in fibrinolysis was obtained in experimental venous and arterial thrombosis models. Incorporation of potato carboxypeptidase inhibitor, a specific TAFI inhibitor, in jugular
⁎ Corresponding author at: Atherothrombosis Laboratory, Center for Applied Medical Research CIMA, Av. Pio XII, 55, 31008 Pamplona, Navarra, Spain. E-mail address: [email protected] (J. Orbe).
vein thrombi resulted in an almost twofold increase in endogenous thrombolysis compared with the control antibody [8]. The profibrinolytic properties of an anti-TAFI monoclonal antibody were also demonstrated in a mouse thromboembolism model [9]. The impact of a carboxypeptidase inhibitor on tPA induced clot lysis was investigated in a rabbit model of arterial thrombosis showing that several parameters associated with the efficiency of thrombolysis were enhanced and the time to reperfusion was reduced [10]. Data from TAFI knockout mice (TAFI−/−) indicates that TAFI deficiency protects against ferric chloride induced vena cava thrombosis [11,12]. However, no differences in the infarct brain area were observed in TAFI −/− compared to controls in another model of ischemic stroke [13]. Circulating microparticles (MPs), small membrane vesicles (≤1 μm) shed by cells in response to various stimuli, have been reported to reflect vascular damage. MPs play a role in thrombosis, inflammation, and endothelial dysfunction but their involvement in cerebrovascular disease remains ill-defined [14]. A variety of studies have demonstrated a role for TAFI in inflammation and cell activation [15] but there are no data on a possible role in the generation of MPs.
http://dx.doi.org/10.1016/j.thromres.2015.06.010 0049-3848/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article as: J. Orbe, et al., Lack of TAFI increases brain damage and microparticle generation after thrombolytic therapy in ischemic stroke, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.010
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We hypothesized that TAFI deficiency favours tPA induced thrombolysis and causes hemorrhagic transformation involving MPs. The aim of this study was to examine the effect of tissue plasminogen activator (tPA) on the rate of intracranial haemorrhage (ICH) and MPs generated in a model of ischemic stroke in TAFI−/− mice. 2. Materials and Methods 2.1. In Vivo Studies 2.1.1. Mouse Model of In Situ Thromboembolic Stroke and Reperfusion The experiments were performed in the following animal groups: TAFI −/− mice with ischemic stroke that received an iv infusion of tPA (10 mg/Kg); and TAFI −/− and WT mice with ischemic stroke infused with a saline buffer. 4 month-old male wild type (WT, C57Bl/6) and TAFI deficient mice (TAFI −/−, C57Bl/6 [16]) were anesthetized with 2.5% isoflurane. A catheter was inserted into the tail vein to allow the intravenous administration of saline (200 μL), or tPA (10 mg/Kg). Thrombin clot formation and assessment of infarct size were performed by thrombin injection (2U/ml) into the middle cerebral artery (MCA), as previously described [17]. Animals were excluded from the study if blood velocity, mesured by laser Doppler Flowmetry, dropped by less than 50%. Experiments were performed in accordance with European Directive 2010/63/EU for the care and use of laboratory animals and approved by the University of Navarra Animal Research Review Committee. 2.1.2. Histological analysis of neuronal degeneration and BBB integrity Mice were humanely killed by CO2 inhalation 24 hours postischemia and perfused with saline. Tissues were then dissected for histological analysis. Brain sections were fixed in 4% paraformaldehyde for 30 min, and degenerating neurons stained with Fluoro-Jade® C (F-J C, Millipore). Fixed brain slides were immersed in a basic alcohol solution (1% NaOH in 80% ethanol) for 5 min, rinsed in 70% ethanol, and distilled water for 2 min, before being incubated in 0.06% KMnO4 solution for 10 min and then stained with a 0.0002% solution of F-J C and 0.0001% DAPI (4',6-diamidino-2-phenylindole) in 0.1% acetic acid. After three rinses in distilled water, sections were air-dried at 50 °C for 5 min or until completely dehydrated. The air-dried slides were then cleared in xylene for at least 1 min and mounted with DPX media (Thermo Scientific). The number of degenerating neurons was determined using an automated Zeiss Axio Imager M1 followed by ImageJ analysis software. All analysis were performed in a blinded fashion. Fixed brain section were rehydrated and permeabilized with 0.2% Trypsin (Sigma) for 15 min. Then, the slides were blocked with 8% BSA in Tris buffer (1 M Tris–HCl, 2.5 M NaCl, pH = 7.5) for 45 min. Double immunofluorescence for CD31 (rat anti-mouse CD31, Dianova) and laminin (rabbit anti-mouse Laminin, Sigma-Aldrich) were performed followed by incubation with peroxidase-labeled secondary IgG (Dako) and amplification with the tyramide signal amplification Plus Cyanine 3 System (Perkin Elmer, Waltham) for CD31 and a goat anti-Rabbit IgG, Alexa Fluor® 488 (Life Technologies) for laminin. 2.1.3. Assessment of the infarct size and haemorrhage Thionine staining was performed on cryostat-cut, coronal brain sections (20 μm). Briefly, sections mounted on slides were dehydrated and stained with thionine (0.125%) for 2 min, dehydrated and mounted with Permount and analyzed with an image analyzer (Image J, National Institutes of Health, US). For volume analysis, one section out of every 10 was stained and lesion (non-stained) areas were measured (covering the entire lesion). To identify the presence of haemorrhage after tPA treatment (if any), a second set of brain sections (20 μm) were stained using the Perls’ Prussian blue method and counterstained with nuclear fast red to reveal iron overload (Hematognost Fe, Merck). Two mice from each treatment were perfused and fixed with paraformaldehyde before embedding in paraffin for DAB (3,3'-diaminobenzidine)
staining. Hematoxylin and eosin (HE) staining was also performed to identify morphological changes. 2.2. In Vitro Studies 2.2.1. Fibrin plate Assay A solution containing 9.5 μM of fibrinogen from human plasma (Sigma) was laid over dishes and clotted by the addition of 20 U human thrombin (Enzyme Research Laboratory). Plates were incubated at 37 °C for 1 h and kept at 4 °C until used. Recombinant tPA (1 U/mL) and plasma euglobulins were pipetted onto the surface of the fibrin plate (n = 5), and further incubated at 37 °C for 24 h. Plasma euglobulins were obtained from pooled plasma (200 μL) diluted 1:10 with distilled water and acidified to pH 5.9 with acetic acid. After 30 min on ice, the precipitate was centrifuged and redissolved in 200 μL HEPES buffer (25 mM, pH 7.5). The lysis areas were determined as a measurement of fibrinolytic activity. 2.2.2. Isolation and purification of microparticles from mouse plasma The blood was drawn from the heart of anaesthetized mice into citrate-anticoagulant syringes (0.129 M, 1:10) and spun-down at 2500 g for 10 min at 4 °C to collect the platelet-poor plasma (PPP) from supernatant. PPP was centrifuged at 13000 g for 2 min at 4 °C to remove residual platelets. The resulting platelet-free plasma (PFP) was centrifuged at 20000 g, for 90 min, at 4 °C to pellet MPs, then washed in HEPES-NaCl buffer (10 mM HEPES, 0.9% NaCl, pH = 7.4) and centrifuged at 20000 g for 90 min. The pellet was then resuspended in HEPES-NaCl buffer pH = 7.4 [18]. 2.2.3. Characterization and quantification of MPs (EMPs, PMPs, LMPs) Purified MPs in suspension (10 μl) were labelled using fluorescein isothiocyanate (FITC)–conjugated Annexin V (for MPs, Invitrogen) and specific monoclonal antibodies: phycoerytrin (PE)-labelled anti-CD31 for endothelium, platelets and leukocytes (BD Pharmingen), PElabelled anti CD41 for platelets (BD Pharmingen) and allophycocyanin (APC)-labelled anti CD45 for leukocytes (BD Pharmingen). The flow cytometry analysis was performed on BD FACSCanto II cytometer (BD Biosciences) after 40 min incubation time (at RT in darkness) as previously described [19]. Isotype control antibodies IgG2a-PE, IgG1-PE, IgG2b-APC were used as negative controls. The gating strategy was based on previous studies [20,21]. Cell origin was studied in Annexin V positive MPs. FlowJo analysis software version 9.3 was used to analyze the results. MP quantification was performed using 10 μl flowcount beads (diameter 3 μm, BD Biosciences) [19]. The number of MPs was calculated using the following formula: [MPs]/ μl = (NMP x1000)/ NB where NMP = total MP counts, NB = total Bead counts and concentration for beads/μl = 10000. 2.2.4. Statistical analysis Data are presented as mean ± SEM. Statistical comparisons were made by Kruskal Wallis and Mann–Whitney U tests, using SPSS version 15.0. A 2-tailed value of p b 0.05 was considered significant. 3. Results 3.1. tPA increases infarct size in TAFI−/− mice The limited data on the role of TAFI as an endogenous fibrinolysis inhibitor in cerebral ischemia prompted us to study the consequences of its ablation in cerebral ischemia upon tPA infusion. In situ injection of thrombin into the MCA resulted in infarct areas restricted to the cortex. As shown in Fig. 1A, tPA treatment significantly increased infarct size in TAFI −/− mice as compared with the control [39.8 ± 14.4 vs 8.9 ± 3.3 mm3, p b 0.05]. Reperfusion was less frequent in saline treated
Please cite this article as: J. Orbe, et al., Lack of TAFI increases brain damage and microparticle generation after thrombolytic therapy in ischemic stroke, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.010
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Fig. 1. tPA treated TAFI−/− mice present increased infarct volume. A) Infarct size (bottom panel) in TAFI−/− mice after thromboembolic stroke model (n = 8-9), * p b 0.05 vs TAFI−/− saline. Upper panel shows representative micrographs of thionine stained slides. B) The fibrinolytic activity of euglobulin plasma samples was determined in baseline WT and TAFI−/− mice by fibrin plate assay. A representative micrograph at 6 h of incubation is shown. C) The lysis was measured at different time points relative to recombinant tPA (1U/mL, Actylise). TAFI−/− samples presented a greater fibrinolytic activity compared to WT n = 4-7. D) Representative micrographs of brain tissues after brain damage. Top line: DAB (left) and Perls' staining (right) illustrate collagenase induced haemorrhage in WT mice. Middle line and bottom line show no staining for DAB or erythrocytes (HE) in saline and tPA infused TAFI−/− mice, respectively.
TAFI−/− animals compared with tPA treated ones (14% vs 62%, n = 8, p = 0.119). A possible explanation for the increased brain damage observed could be related to an excessive fibrinolytic activity associated with the absence of TAFI. Plasma euglobulins from TAFI −/− and WT mice were assayed on fibrin plates. As shown in Fig. 1B-C, TAFI−/− plasmas presented measurable lysis areas at 4, 6, 8 and 24 h after incubation, while WT samples only displayed fibrinolytic activity from 8 h onwards. Since increased fibrinolytic activity could lead to haemorrhagic complications in ischemic stroke, we determined the possible involvement of TAFI deficiency in the haemorrhagic complications related to tPA treatment in brain sections. Neither Perls’ nor DAB staining showed any evidence of ICH under these experimental conditions in any group, as compared with positive controls performed with collagenase type VII (Fig. 1D). 3.2. tPA increases neuronal degeneration in TAFI−/− mice In order to understand possible mechanisms for increased infarct size in TAFI −/−, we stained brain sections with F-J C as a marker of neuronal degeneration. F-J C+ cells were localized in the infarct area and increased numbers were observed in tPA-treated TAFI −/− mice compared to untreated null mice (Fig. 2), suggesting a higher
susceptibility to tissue damage and neuronal degeneration after tPA infusion in the absence of TAFI. To address whether the later could be related to BBB defects in TAFI −/− mice in normal conditions, we performed immunofluorescence for laminin (basal lamina component) and CD31 (endothelial marker) in brain sections of both genotypes at baseline. As shown in Fig. 2C, no differences in the fluorescent signal were detected between WT and TAFI−/− mice. These results suggest that TAFI might play a role in pathological conditions, but not in physiological conditions, as TAFI−/− mice are perfectly viable and only display abnormal phenotypes when challenged [22,23]. 3.3. TAFI ablation increases the number of circulating MPs in vivo MPs have been regarded as carriers of different bioactive molecules that can influence cell behaviour at local or distant places in physiological and pathological conditions. We studied the number of circulating MPs by flow cytometry in plasma of TAFI −/− and WT mice before and after thromboembolic stroke. As shown in Fig. 3A, the number of MPs assessed by 3 μm flowcount beads (Fig. 3B) was similar between the genotypes at baseline (102 ± 37 MPs/μL WT vs 158 ± 35 MPs/μL TAFI −/−). After ischemia, the number of circulating MPs was greatly increased in saline (395 ± 67 MPs/μL) or tPA-infused TAFI deficient mice (375 ± 60 MPs/μL), while no increase was reported in saline
Please cite this article as: J. Orbe, et al., Lack of TAFI increases brain damage and microparticle generation after thrombolytic therapy in ischemic stroke, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.010
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Fig. 2. tPA treated TAFI−/− mice present increased neuronal degeneration. A) Number of degenerating neurons (F-J C staining) in TAFI−/− mice after stroke (n = 5, p = 0.08). B) Representative micrographs of F-J C stained brain sections. Degenerated neurons appear in green. Scale bar denotes 10 μm. C) Immunofluorescence for laminin and CD31 in WT and TAFI−/− brain slides at baseline. Green: laminin, red: CD31, blue: DAPI. Scale bar denotes 50 μm.
(193 ± 27 MPs/μL) or tPA-infused (118 ± 35 MPs/μL) ischemic WT mice. Regarding the origin of circulating MPs after MCA occlusion, we found a significant increase of endothelial (AnnV+CD31+MPs) and platelet derived MPs (AnnV+CD41+MPs) in TAFI−/− mice compared to WT, regardless of the thrombolytic therapy. Inflammatory cell derived MPs (AnnV+CD45+MPs) decreased after ischemic stroke in both genotypes compared to baseline (Fig. 4). 4. Discussion This study demonstrates that TAFI deficiency results in increased brain damage in a model of thrombolysis after ischemic stroke. Previous studies have reported that neither the genetic deficiency of TAFI nor its pharmacological inhibition succeeded in reducing infarct size, occlusion time or microhaemorrhage presence after thromboembolic stroke or venous injury [13,24], but there are still no data regarding the effect of the thrombolytic therapy with tPA in TAFI−/− mice. Early treatment of WT mice with the standard dose of tPA (10 mg/Kg) has been associated with a significant reduction in lesion volume compared to saline in the current thromboembolic model [17,20]. Quite surprisingly, here we report that tPA aggravated brain ischemic lesion in the absence of TAFI. Tissue damage depends on fibrinolytic capacity and excessive fibrinolytic activity could compromise stroke evolution. Our results reporting increased fibrinolytic activity in TAFI−/− mice agree with previous data showing that TAFI deficiency in mice results in enhanced clot lysis as assessed in whole blood by thromboelastographic analysis [25]. Although hyperfibrinolysis could promote bleeding, no evidence of ICH under these experimental conditions was observed in any group. It is worthwhile mentioning that the
brain microvasculature demonstrates a remarkably consistent pattern of structural and functional organization that offers an unusual degree of protection against haemorrhage [26] and TAFI−/− mice presented similar vessel integrity to WT in healthy brain. On the other hand, although TAFI is mainly identified as a down-regulator of fibrinolysis, recent studies have reported new roles for TAFI as an anti-inflammatory and endothelial barrier stabilizing agent [15,27] that might contribute to explaining the absence of brain haemorrhage in this model. Our results also indicate a higher susceptibility to tissue damage and neuronal degeneration after tPA infusion in the absence of TAFI. The role of tPA in this field is controversial [28] and several studies have shown the deleterious effect of tPA in cerebral parenchyma, but whether TAFI depletion could further contribute to tPA-induced neurotoxicity will require more investigation. TAFI is not only a regulator of fibrinolysis, but it is also involved in the regulation of inflammation leading to an enhanced inflammatory response in TAFI −/− mouse models [29,30]. It is well known that a number of stress conditions and inflammatory mediators may stimulate cell activation and MP production [31], but there are no data regarding the role of TAFI on MP generation. Here, we showed for the first time, that after ischemia the number of circulating MPs was greatly increased in TAFI deficient mice, but not in WT. This phenomenon was observed regardless of the presence of tPA in TAFI −/− mice. The thrombin injected to induce ischemia might be responsible for the increase in MPs, however our results showing no differences in MP counts in ischemic WT mice compared to baseline, suggest that TAFI deficiency might be responsible for increased MP release, rather than thrombin injection. Based on previous reports on membrane blebbing and MPs release induced by plasmin [18,32], we propose that higher fibrinolytic activity
Please cite this article as: J. Orbe, et al., Lack of TAFI increases brain damage and microparticle generation after thrombolytic therapy in ischemic stroke, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.010
J. Orbe et al. / Thrombosis Research xxx (2015) xxx–xxx
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we only consider the number of MPs that are double positive for Annexin V and the specific cell marker. Recent studies have reported that in contrast to the classical theory of extracellular vesicle (EV) formation where the majority of EVs should be Annexin V positive, most of them do not expose phosphatidylserine. This is the case for platelet MPs [33], showing that only 30% of them were double positive. Increased release of MPs has been described as being associated with the acute or active phases of several neurological disorders [34]. Furthermore, the number of MPs derived from endothelial cells or platelets have been linked to the extent of cerebral ischemia by several groups [35] and associated with the pro-thrombotic state leading to stroke. TAFI −/− mice appear to have a normal phenotype until challenged and the increase in MP generation might suggest a higher inflammatory status in these mice [15,27] leading to the generation of MPs after stroke, which could act in a paracrine fashion, promoting further vascular inflammation [36], thus increasing ischemic lesions. We conclude that TAFI deficiency results in increased brain damage in a model of thrombolysis after ischemic stroke, which is associated with neuronal degeneration and MP production.
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Authors contribution Study conception and design: Orbe J and Paramo JA. Acquisition of data: Rodriguez JA, Belzunce M, Alexandru N, Bibiot P and Meijers JCM. Analysis and interpretation of data: Roncal C, Orbe J, Alexandru N and Georgescu A. Drafting of manuscript: Orbe J, Roncal C and Rodriguez JA. Critical revision: Georgescu A, Meijers JCM and Paramo JA.
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Fig. 3. MPs are increased in TAFI deficient mice. A) MP counts at baseline and 24 hours after stroke in WT and TAFI −/− mice. ** p b 0.01 vs WT, $ p b 0.05 $$ p b 0.01 vs TAFI−/− baseline. B) Representative dot-plot showing the quantification strategy based on 3 μm calibrated size beads.
found in TAFI−/− mice could be implicated in MPs increase observed in these mice. Moreover, it could be the result of at least three additional factors: increase in circulating MPs caused by cerebrovascular ischemia, cell membrane shedding induced by inflammation, and the lack of the antiinflammatory effects promoted by TAFI [29,31]. Our analysis regarding the cell type origin identified endothelial (CD31+MPs) and platelet derived MPs (CD41+MPs) as those that are significantly augmented in TAFI −/− mice. More extensive studies should be performed in order to confirm those data, as in this study % of double positive MPs/ Total MP numbers
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Fig. 4. Annexin V positive MPs of Endothelial origin (AnnV+CD31+MPs) and platelet origin (AnnV+CD41+MPs) are augmented in TAFI−/− mice. The cellular origin of circulating MPs was analyzed by flow cytometry in (n = 6-8). * p b 0.05 vs WT and $ p b 0.05; $$ p b 0.01 $$$ p b 0.001 vs TAFI−/−.
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Please cite this article as: J. Orbe, et al., Lack of TAFI increases brain damage and microparticle generation after thrombolytic therapy in ischemic stroke, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.010
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Lack of TAFI increases brain damage and microparticle generation after thrombolytic therapy in ischemic stroke J. Orbe a,⁎, N. Alexandru b, C. Roncal a, M. Belzunce a, P. Bibiot a, J.A. Rodriguez a, J.C.M. Meijers c,d, A. Georgescu b, J.A. Paramo a a
Laboratory of Atherothrombosis, Program of Cardiovascular Diseases, CIMA-University of Navarra, Navarra Institute for Health Research (IdiSNA), Pamplona, Spain Pathophysiology and Pharmacology Department, Institute of Cellular Biology and Pathology ‘Nicolae Simionescu’ of Romanian Academy, Bucharest, Romania Experimental Vascular Medicine, Academic Medical Center, University of Amsterdam, the Netherlands d Department of Plasma Proteins, Sanquin Research, Amsterdam, the Netherlands b c
a r t i c l e
i n f o
Article history: Received 18 February 2015 Received in revised form 29 May 2015 Accepted 8 June 2015 Available online xxxx Keywords: TAFI Fibrinolysis Thrombolytic therapy Circulating cell-derived microparticles Inflammation
a b s t r a c t Background: Thrombin-activatable fibrinolysis inhibitor (TAFI) plays an important role in coagulation and fibrinolysis. Whereas TAFI deficiency may lead to a haemorrhagic tendency, data from TAFI knockout mice (TAFI −/−) are controversial and no differences have been reported in these animals after ischemic stroke. There are also no data regarding the role of circulating microparticles (MPs) in TAFI−/−. Objectives: to examine the effect of tPA on the rate of intracranial haemorrhage (ICH) and on MPs generated in a model of ischemic stroke in TAFI−/− mice. Methods: Thrombin was injected into the middle cerebral artery (MCA) to analyse the effect of tPA (10 mg/Kg) on the infarct size and haemorrhage in the absence of TAFI. Immunofluorescence for Fluoro-Jade C was performed on frozen brain slides to analyse neuronal degeneration after ischemia. MPs were isolated from mouse blood and their concentrations calculated by flow cytometry. Results: Compared with saline, tPA significantly increased the infarct size in TAFI−/− mice (p b 0.05). Although plasma fibrinolytic activity (fibrin plate assay) was higher in these animals, no macroscopic or microscopic ICH was detected. A positive signal for apoptosis and degenerating neurons was observed in the infarct area, being significantly higher in tPA treated TAFI−/− mice (p b 0.05). Interestingly, higher numbers of MPs were found in TAFI−/− plasma as compared to wild type, after stroke (p b 0.05). Conclusions: TAFI deficiency results in increased brain damage in a model of thrombolysis after ischemic stroke, which was not associated with bleeding but with neuronal degeneration and MP production. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma procarboxypeptidase that upon activation by thrombin or thrombin/ thrombomodulin turns into a potent antifibrinolytic enzyme [1–3]. The activation of TAFI by thrombin implies that the coagulation system plays a role in the regulation of fibrinolysis and that disturbances in TAFI levels will affect the rate of lysis [4–6]. Since clot stabilisation seems to be a requisite for the prevention of bleeding, it could be assumed that TAFI deficiency would lead to a hemorrhagic tendency by inducing premature fibrinolysis [7]. In vivo evidence for the role of TAFI in fibrinolysis was obtained in experimental venous and arterial thrombosis models. Incorporation of potato carboxypeptidase inhibitor, a specific TAFI inhibitor, in jugular
⁎ Corresponding author at: Atherothrombosis Laboratory, Center for Applied Medical Research CIMA, Av. Pio XII, 55, 31008 Pamplona, Navarra, Spain. E-mail address: [email protected] (J. Orbe).
vein thrombi resulted in an almost twofold increase in endogenous thrombolysis compared with the control antibody [8]. The profibrinolytic properties of an anti-TAFI monoclonal antibody were also demonstrated in a mouse thromboembolism model [9]. The impact of a carboxypeptidase inhibitor on tPA induced clot lysis was investigated in a rabbit model of arterial thrombosis showing that several parameters associated with the efficiency of thrombolysis were enhanced and the time to reperfusion was reduced [10]. Data from TAFI knockout mice (TAFI−/−) indicates that TAFI deficiency protects against ferric chloride induced vena cava thrombosis [11,12]. However, no differences in the infarct brain area were observed in TAFI −/− compared to controls in another model of ischemic stroke [13]. Circulating microparticles (MPs), small membrane vesicles (≤1 μm) shed by cells in response to various stimuli, have been reported to reflect vascular damage. MPs play a role in thrombosis, inflammation, and endothelial dysfunction but their involvement in cerebrovascular disease remains ill-defined [14]. A variety of studies have demonstrated a role for TAFI in inflammation and cell activation [15] but there are no data on a possible role in the generation of MPs.
http://dx.doi.org/10.1016/j.thromres.2015.06.010 0049-3848/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article as: J. Orbe, et al., Lack of TAFI increases brain damage and microparticle generation after thrombolytic therapy in ischemic stroke, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.010
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We hypothesized that TAFI deficiency favours tPA induced thrombolysis and causes hemorrhagic transformation involving MPs. The aim of this study was to examine the effect of tissue plasminogen activator (tPA) on the rate of intracranial haemorrhage (ICH) and MPs generated in a model of ischemic stroke in TAFI−/− mice. 2. Materials and Methods 2.1. In Vivo Studies 2.1.1. Mouse Model of In Situ Thromboembolic Stroke and Reperfusion The experiments were performed in the following animal groups: TAFI −/− mice with ischemic stroke that received an iv infusion of tPA (10 mg/Kg); and TAFI −/− and WT mice with ischemic stroke infused with a saline buffer. 4 month-old male wild type (WT, C57Bl/6) and TAFI deficient mice (TAFI −/−, C57Bl/6 [16]) were anesthetized with 2.5% isoflurane. A catheter was inserted into the tail vein to allow the intravenous administration of saline (200 μL), or tPA (10 mg/Kg). Thrombin clot formation and assessment of infarct size were performed by thrombin injection (2U/ml) into the middle cerebral artery (MCA), as previously described [17]. Animals were excluded from the study if blood velocity, mesured by laser Doppler Flowmetry, dropped by less than 50%. Experiments were performed in accordance with European Directive 2010/63/EU for the care and use of laboratory animals and approved by the University of Navarra Animal Research Review Committee. 2.1.2. Histological analysis of neuronal degeneration and BBB integrity Mice were humanely killed by CO2 inhalation 24 hours postischemia and perfused with saline. Tissues were then dissected for histological analysis. Brain sections were fixed in 4% paraformaldehyde for 30 min, and degenerating neurons stained with Fluoro-Jade® C (F-J C, Millipore). Fixed brain slides were immersed in a basic alcohol solution (1% NaOH in 80% ethanol) for 5 min, rinsed in 70% ethanol, and distilled water for 2 min, before being incubated in 0.06% KMnO4 solution for 10 min and then stained with a 0.0002% solution of F-J C and 0.0001% DAPI (4',6-diamidino-2-phenylindole) in 0.1% acetic acid. After three rinses in distilled water, sections were air-dried at 50 °C for 5 min or until completely dehydrated. The air-dried slides were then cleared in xylene for at least 1 min and mounted with DPX media (Thermo Scientific). The number of degenerating neurons was determined using an automated Zeiss Axio Imager M1 followed by ImageJ analysis software. All analysis were performed in a blinded fashion. Fixed brain section were rehydrated and permeabilized with 0.2% Trypsin (Sigma) for 15 min. Then, the slides were blocked with 8% BSA in Tris buffer (1 M Tris–HCl, 2.5 M NaCl, pH = 7.5) for 45 min. Double immunofluorescence for CD31 (rat anti-mouse CD31, Dianova) and laminin (rabbit anti-mouse Laminin, Sigma-Aldrich) were performed followed by incubation with peroxidase-labeled secondary IgG (Dako) and amplification with the tyramide signal amplification Plus Cyanine 3 System (Perkin Elmer, Waltham) for CD31 and a goat anti-Rabbit IgG, Alexa Fluor® 488 (Life Technologies) for laminin. 2.1.3. Assessment of the infarct size and haemorrhage Thionine staining was performed on cryostat-cut, coronal brain sections (20 μm). Briefly, sections mounted on slides were dehydrated and stained with thionine (0.125%) for 2 min, dehydrated and mounted with Permount and analyzed with an image analyzer (Image J, National Institutes of Health, US). For volume analysis, one section out of every 10 was stained and lesion (non-stained) areas were measured (covering the entire lesion). To identify the presence of haemorrhage after tPA treatment (if any), a second set of brain sections (20 μm) were stained using the Perls’ Prussian blue method and counterstained with nuclear fast red to reveal iron overload (Hematognost Fe, Merck). Two mice from each treatment were perfused and fixed with paraformaldehyde before embedding in paraffin for DAB (3,3'-diaminobenzidine)
staining. Hematoxylin and eosin (HE) staining was also performed to identify morphological changes. 2.2. In Vitro Studies 2.2.1. Fibrin plate Assay A solution containing 9.5 μM of fibrinogen from human plasma (Sigma) was laid over dishes and clotted by the addition of 20 U human thrombin (Enzyme Research Laboratory). Plates were incubated at 37 °C for 1 h and kept at 4 °C until used. Recombinant tPA (1 U/mL) and plasma euglobulins were pipetted onto the surface of the fibrin plate (n = 5), and further incubated at 37 °C for 24 h. Plasma euglobulins were obtained from pooled plasma (200 μL) diluted 1:10 with distilled water and acidified to pH 5.9 with acetic acid. After 30 min on ice, the precipitate was centrifuged and redissolved in 200 μL HEPES buffer (25 mM, pH 7.5). The lysis areas were determined as a measurement of fibrinolytic activity. 2.2.2. Isolation and purification of microparticles from mouse plasma The blood was drawn from the heart of anaesthetized mice into citrate-anticoagulant syringes (0.129 M, 1:10) and spun-down at 2500 g for 10 min at 4 °C to collect the platelet-poor plasma (PPP) from supernatant. PPP was centrifuged at 13000 g for 2 min at 4 °C to remove residual platelets. The resulting platelet-free plasma (PFP) was centrifuged at 20000 g, for 90 min, at 4 °C to pellet MPs, then washed in HEPES-NaCl buffer (10 mM HEPES, 0.9% NaCl, pH = 7.4) and centrifuged at 20000 g for 90 min. The pellet was then resuspended in HEPES-NaCl buffer pH = 7.4 [18]. 2.2.3. Characterization and quantification of MPs (EMPs, PMPs, LMPs) Purified MPs in suspension (10 μl) were labelled using fluorescein isothiocyanate (FITC)–conjugated Annexin V (for MPs, Invitrogen) and specific monoclonal antibodies: phycoerytrin (PE)-labelled anti-CD31 for endothelium, platelets and leukocytes (BD Pharmingen), PElabelled anti CD41 for platelets (BD Pharmingen) and allophycocyanin (APC)-labelled anti CD45 for leukocytes (BD Pharmingen). The flow cytometry analysis was performed on BD FACSCanto II cytometer (BD Biosciences) after 40 min incubation time (at RT in darkness) as previously described [19]. Isotype control antibodies IgG2a-PE, IgG1-PE, IgG2b-APC were used as negative controls. The gating strategy was based on previous studies [20,21]. Cell origin was studied in Annexin V positive MPs. FlowJo analysis software version 9.3 was used to analyze the results. MP quantification was performed using 10 μl flowcount beads (diameter 3 μm, BD Biosciences) [19]. The number of MPs was calculated using the following formula: [MPs]/ μl = (NMP x1000)/ NB where NMP = total MP counts, NB = total Bead counts and concentration for beads/μl = 10000. 2.2.4. Statistical analysis Data are presented as mean ± SEM. Statistical comparisons were made by Kruskal Wallis and Mann–Whitney U tests, using SPSS version 15.0. A 2-tailed value of p b 0.05 was considered significant. 3. Results 3.1. tPA increases infarct size in TAFI−/− mice The limited data on the role of TAFI as an endogenous fibrinolysis inhibitor in cerebral ischemia prompted us to study the consequences of its ablation in cerebral ischemia upon tPA infusion. In situ injection of thrombin into the MCA resulted in infarct areas restricted to the cortex. As shown in Fig. 1A, tPA treatment significantly increased infarct size in TAFI −/− mice as compared with the control [39.8 ± 14.4 vs 8.9 ± 3.3 mm3, p b 0.05]. Reperfusion was less frequent in saline treated
Please cite this article as: J. Orbe, et al., Lack of TAFI increases brain damage and microparticle generation after thrombolytic therapy in ischemic stroke, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.010
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Fig. 1. tPA treated TAFI−/− mice present increased infarct volume. A) Infarct size (bottom panel) in TAFI−/− mice after thromboembolic stroke model (n = 8-9), * p b 0.05 vs TAFI−/− saline. Upper panel shows representative micrographs of thionine stained slides. B) The fibrinolytic activity of euglobulin plasma samples was determined in baseline WT and TAFI−/− mice by fibrin plate assay. A representative micrograph at 6 h of incubation is shown. C) The lysis was measured at different time points relative to recombinant tPA (1U/mL, Actylise). TAFI−/− samples presented a greater fibrinolytic activity compared to WT n = 4-7. D) Representative micrographs of brain tissues after brain damage. Top line: DAB (left) and Perls' staining (right) illustrate collagenase induced haemorrhage in WT mice. Middle line and bottom line show no staining for DAB or erythrocytes (HE) in saline and tPA infused TAFI−/− mice, respectively.
TAFI−/− animals compared with tPA treated ones (14% vs 62%, n = 8, p = 0.119). A possible explanation for the increased brain damage observed could be related to an excessive fibrinolytic activity associated with the absence of TAFI. Plasma euglobulins from TAFI −/− and WT mice were assayed on fibrin plates. As shown in Fig. 1B-C, TAFI−/− plasmas presented measurable lysis areas at 4, 6, 8 and 24 h after incubation, while WT samples only displayed fibrinolytic activity from 8 h onwards. Since increased fibrinolytic activity could lead to haemorrhagic complications in ischemic stroke, we determined the possible involvement of TAFI deficiency in the haemorrhagic complications related to tPA treatment in brain sections. Neither Perls’ nor DAB staining showed any evidence of ICH under these experimental conditions in any group, as compared with positive controls performed with collagenase type VII (Fig. 1D). 3.2. tPA increases neuronal degeneration in TAFI−/− mice In order to understand possible mechanisms for increased infarct size in TAFI −/−, we stained brain sections with F-J C as a marker of neuronal degeneration. F-J C+ cells were localized in the infarct area and increased numbers were observed in tPA-treated TAFI −/− mice compared to untreated null mice (Fig. 2), suggesting a higher
susceptibility to tissue damage and neuronal degeneration after tPA infusion in the absence of TAFI. To address whether the later could be related to BBB defects in TAFI −/− mice in normal conditions, we performed immunofluorescence for laminin (basal lamina component) and CD31 (endothelial marker) in brain sections of both genotypes at baseline. As shown in Fig. 2C, no differences in the fluorescent signal were detected between WT and TAFI−/− mice. These results suggest that TAFI might play a role in pathological conditions, but not in physiological conditions, as TAFI−/− mice are perfectly viable and only display abnormal phenotypes when challenged [22,23]. 3.3. TAFI ablation increases the number of circulating MPs in vivo MPs have been regarded as carriers of different bioactive molecules that can influence cell behaviour at local or distant places in physiological and pathological conditions. We studied the number of circulating MPs by flow cytometry in plasma of TAFI −/− and WT mice before and after thromboembolic stroke. As shown in Fig. 3A, the number of MPs assessed by 3 μm flowcount beads (Fig. 3B) was similar between the genotypes at baseline (102 ± 37 MPs/μL WT vs 158 ± 35 MPs/μL TAFI −/−). After ischemia, the number of circulating MPs was greatly increased in saline (395 ± 67 MPs/μL) or tPA-infused TAFI deficient mice (375 ± 60 MPs/μL), while no increase was reported in saline
Please cite this article as: J. Orbe, et al., Lack of TAFI increases brain damage and microparticle generation after thrombolytic therapy in ischemic stroke, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.010
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Fig. 2. tPA treated TAFI−/− mice present increased neuronal degeneration. A) Number of degenerating neurons (F-J C staining) in TAFI−/− mice after stroke (n = 5, p = 0.08). B) Representative micrographs of F-J C stained brain sections. Degenerated neurons appear in green. Scale bar denotes 10 μm. C) Immunofluorescence for laminin and CD31 in WT and TAFI−/− brain slides at baseline. Green: laminin, red: CD31, blue: DAPI. Scale bar denotes 50 μm.
(193 ± 27 MPs/μL) or tPA-infused (118 ± 35 MPs/μL) ischemic WT mice. Regarding the origin of circulating MPs after MCA occlusion, we found a significant increase of endothelial (AnnV+CD31+MPs) and platelet derived MPs (AnnV+CD41+MPs) in TAFI−/− mice compared to WT, regardless of the thrombolytic therapy. Inflammatory cell derived MPs (AnnV+CD45+MPs) decreased after ischemic stroke in both genotypes compared to baseline (Fig. 4). 4. Discussion This study demonstrates that TAFI deficiency results in increased brain damage in a model of thrombolysis after ischemic stroke. Previous studies have reported that neither the genetic deficiency of TAFI nor its pharmacological inhibition succeeded in reducing infarct size, occlusion time or microhaemorrhage presence after thromboembolic stroke or venous injury [13,24], but there are still no data regarding the effect of the thrombolytic therapy with tPA in TAFI−/− mice. Early treatment of WT mice with the standard dose of tPA (10 mg/Kg) has been associated with a significant reduction in lesion volume compared to saline in the current thromboembolic model [17,20]. Quite surprisingly, here we report that tPA aggravated brain ischemic lesion in the absence of TAFI. Tissue damage depends on fibrinolytic capacity and excessive fibrinolytic activity could compromise stroke evolution. Our results reporting increased fibrinolytic activity in TAFI−/− mice agree with previous data showing that TAFI deficiency in mice results in enhanced clot lysis as assessed in whole blood by thromboelastographic analysis [25]. Although hyperfibrinolysis could promote bleeding, no evidence of ICH under these experimental conditions was observed in any group. It is worthwhile mentioning that the
brain microvasculature demonstrates a remarkably consistent pattern of structural and functional organization that offers an unusual degree of protection against haemorrhage [26] and TAFI−/− mice presented similar vessel integrity to WT in healthy brain. On the other hand, although TAFI is mainly identified as a down-regulator of fibrinolysis, recent studies have reported new roles for TAFI as an anti-inflammatory and endothelial barrier stabilizing agent [15,27] that might contribute to explaining the absence of brain haemorrhage in this model. Our results also indicate a higher susceptibility to tissue damage and neuronal degeneration after tPA infusion in the absence of TAFI. The role of tPA in this field is controversial [28] and several studies have shown the deleterious effect of tPA in cerebral parenchyma, but whether TAFI depletion could further contribute to tPA-induced neurotoxicity will require more investigation. TAFI is not only a regulator of fibrinolysis, but it is also involved in the regulation of inflammation leading to an enhanced inflammatory response in TAFI −/− mouse models [29,30]. It is well known that a number of stress conditions and inflammatory mediators may stimulate cell activation and MP production [31], but there are no data regarding the role of TAFI on MP generation. Here, we showed for the first time, that after ischemia the number of circulating MPs was greatly increased in TAFI deficient mice, but not in WT. This phenomenon was observed regardless of the presence of tPA in TAFI −/− mice. The thrombin injected to induce ischemia might be responsible for the increase in MPs, however our results showing no differences in MP counts in ischemic WT mice compared to baseline, suggest that TAFI deficiency might be responsible for increased MP release, rather than thrombin injection. Based on previous reports on membrane blebbing and MPs release induced by plasmin [18,32], we propose that higher fibrinolytic activity
Please cite this article as: J. Orbe, et al., Lack of TAFI increases brain damage and microparticle generation after thrombolytic therapy in ischemic stroke, Thromb Res (2015), http://dx.doi.org/10.1016/j.thromres.2015.06.010
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we only consider the number of MPs that are double positive for Annexin V and the specific cell marker. Recent studies have reported that in contrast to the classical theory of extracellular vesicle (EV) formation where the majority of EVs should be Annexin V positive, most of them do not expose phosphatidylserine. This is the case for platelet MPs [33], showing that only 30% of them were double positive. Increased release of MPs has been described as being associated with the acute or active phases of several neurological disorders [34]. Furthermore, the number of MPs derived from endothelial cells or platelets have been linked to the extent of cerebral ischemia by several groups [35] and associated with the pro-thrombotic state leading to stroke. TAFI −/− mice appear to have a normal phenotype until challenged and the increase in MP generation might suggest a higher inflammatory status in these mice [15,27] leading to the generation of MPs after stroke, which could act in a paracrine fashion, promoting further vascular inflammation [36], thus increasing ischemic lesions. We conclude that TAFI deficiency results in increased brain damage in a model of thrombolysis after ischemic stroke, which is associated with neuronal degeneration and MP production.
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Authors contribution Study conception and design: Orbe J and Paramo JA. Acquisition of data: Rodriguez JA, Belzunce M, Alexandru N, Bibiot P and Meijers JCM. Analysis and interpretation of data: Roncal C, Orbe J, Alexandru N and Georgescu A. Drafting of manuscript: Orbe J, Roncal C and Rodriguez JA. Critical revision: Georgescu A, Meijers JCM and Paramo JA.
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Conflict of Interest
Fig. 3. MPs are increased in TAFI deficient mice. A) MP counts at baseline and 24 hours after stroke in WT and TAFI −/− mice. ** p b 0.01 vs WT, $ p b 0.05 $$ p b 0.01 vs TAFI−/− baseline. B) Representative dot-plot showing the quantification strategy based on 3 μm calibrated size beads.
found in TAFI−/− mice could be implicated in MPs increase observed in these mice. Moreover, it could be the result of at least three additional factors: increase in circulating MPs caused by cerebrovascular ischemia, cell membrane shedding induced by inflammation, and the lack of the antiinflammatory effects promoted by TAFI [29,31]. Our analysis regarding the cell type origin identified endothelial (CD31+MPs) and platelet derived MPs (CD41+MPs) as those that are significantly augmented in TAFI −/− mice. More extensive studies should be performed in order to confirm those data, as in this study % of double positive MPs/ Total MP numbers
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CD45
Fig. 4. Annexin V positive MPs of Endothelial origin (AnnV+CD31+MPs) and platelet origin (AnnV+CD41+MPs) are augmented in TAFI−/− mice. The cellular origin of circulating MPs was analyzed by flow cytometry in (n = 6-8). * p b 0.05 vs WT and $ p b 0.05; $$ p b 0.01 $$$ p b 0.001 vs TAFI−/−.
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