OBJECTIVE:We aimed to analyze ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) in relation to arterial recanalization in patients treated with IV tissue plasminogen activator (tPA) and in relation to futile recanalization in patients treated with mechanical thrombectomy.METHODS:Acute ischemic stroke patients (n = 108) with documented arterial occlusions treated with IV-tPA were selected. ADAMTS13 activity was measured by ELISA in samples collected before treatment. Recanalization was assessed at 2 hours by transcranial Doppler. In 78 consecutive patients treated with endovascular thrombectomy, ADAMTS13 antigen was measured by ELISA and futile recanalization was defined as complete recanalization plus modified Rankin Scale score >2 at 3 months. Independent predictors of recanalization and futile recanalization were determined by logistic regression, adjusted by age, NIH Stroke Scale score, and time from stroke onset.RESULTS:Patients who achieved tPA-induced recanalization had higher baseline ADAMTS13 activity (78.1% [68%-88%] vs 70.1% [61%-79%], p = 0.021). In logistic regression analysis, ADAMTS13 activity >75% was an independent predictor of recanalization (odds ratio = 6.76 [1.52-30.02], p = 0.012), together with absence of early ischemic signs and Oxfordshire Community Stroke Project classification. Regarding endovascular therapies, a reduced ADAMTS13 concentration (<982 ng/mL) was an independent predictor of futile recanalization (odds ratio = 67.4 [1.4-3,282.1], p = 0.034), together with age and diabetes mellitus. The addition of ADAMTS13 to clinical predictors of tPA-induced recanalization and futile recanalization improved discrimination and reclassification (integrated discrimination improvement = 10.06% and 28.4%, net reclassification improvement = 61.0% and 107.4%, respectively).CONCLUSIONS:A reduced ADAMTS13 was associated with poor response to recanalization therapies. If confirmed in future prospective studies, a panel of blood biomarkers including ADAMTS13 might be a useful tool to guide reperfusion therapies.

AIM:We report the study of a familial rare disease with recurrent venous thromboembolic events that remained undiagnosed for many years using standard coagulation and hemostasis techniques.METHODS:Exome sequencing was performed in three familial cases with venous thromboembolic disease and one familial control using NimbleGen exome array. Clot lysis experiments were performed to analyze the reasons of the altered fibrinolytic activity caused by the mutation found.RESULTS:We found a mutation that consists of a R458C substitution on the fibrinogen alpha chain (FGA) gene confirmed in 13 new familial subjects that causes a rare subtype of dysfibrinogenemia characterized by venous thromboembolic events. The mutation was already reported to be associated with a fibrinogen variant called fibrinogen Bordeaux. Clot-lysis experiments showed a decreased and slower fibrinolytic activity in carriers of this mutation as compared to normal subjects, thus demonstrating an impaired fibrinolysis of fibrinogen Bordeaux.CONCLUSIONS:The exome sequencing and clot-lysis experiments might be powerful tools to diagnose idiopathic thrombophilias after an unsuccessful set of biochemical laboratory tests. Fibrinogen Bordeaux is associated with impaired fibrinolysis in this family with idiopathic thrombophilia.

INTRODUCTION: Biological markers predicting tPA response in acute stroke could be used for dose adjustments or early selection of patients for endovascular procedures. ADAMTS13 (A Disintegrin And Metalloproteinase with a ThromboSpondin type 1 motif, member 13) inactivates Von Willebrand Factor by cleaving it, and its deficiency may generate prothrombotic diseases such as thrombotic thrombocytopenic purpura. We aimed to analyze ADAMTS13 activity in acute stroke patients and its relation to vessel patency among those treated with intravenous tPA. METHODS: Acute ischemic stroke patients (n=104) with documented arterial occlusion by transcranial Doppler (TCD), who received tPA within the first 4.5 hours after symptoms onset were recruited and compared with 38 age-matched healthy subjects. Samples were collected at baseline, before thrombolytic treatment, and ADAMTS13 activity was measured by ELISA and expressed as %. A temporal profile of ADAMTS13 activity was determined at 24 hours and 3 months in a subset of 10 patients. Recanalization was assessed 2 hours after tPA bolus by TCD, using thrombolysis in brain ischemia (TIBI) flow grading system. Logistic regression analysis was conducted to determine independent predictors of 2-hour recanalization in patients with proximal arterial occlusions. RESULTS: ADAMTS13 activity was consistently lower in stroke patients than in healthy controls (p<0.001), and remained lower at 24 hours and 3 months. For those patients who presented arterial recanalization at 2 hours, higher baseline ADAMTS13 activity (p=0.032) was noted. In logistic regression analysis from 72 patients with proximal MCA occlusion, ADAMTS13 activity >74.72% was an independent predictor of recanalization [OR=5.148 (1.463-18.111), p=0.011], together with early ischemic signs at baseline neuroimaging [OR=0.065 (0.006-0.712), p=0.025] and OCSP classification (TACI vs. PACI) [OR=0.072 (0.011-0.45), p=0.005]. CONCLUSIONS: In stroke patients treated with tPA, ADAMTS13 activity may be used to monitor the treatment as well as to make decisions regarding more aggressive reperfusion therapies when the absence of a response to intravenous tPA is anticipated.

A rapid differentiation of acute ischemic stroke and intracerebral hemorrhage (ICH) is essential for an adequate treatment and to promote a better outcome. Our aim was to identify new plasma biomarkers to differentiate stroke subtypes and to combine their diagnostic ability with other biomarkers already described for this clinical indication. Plasma samples of ischemic stroke patients (36) and ICH patients (10) were screened using a 177 antibodies library, and 11 showed different concentrations among stroke subtypes (p < 0.05), mainly chemokines, growth factors and angiogenic factors. Five proteins were selected for replication in 16 ischemic stroke patients and 16 ICH patients, and retinol-binding protein 4 (RPB4), apolipoprotein B100 and pigment epithelial-derived factor were replicated (p < 0.05). These proteins, together with glial fibrillary acidic protein (GFAP) and receptor for advanced glycation end product, were tested in 38 ischemic stroke and 28 ICH samples. Finally, RBP4 >61 μg/mL and GFAP <0.07 ng/mL showed a specificity of 100% for both subtypes. Moreover, after multivariate logistic regression analysis, RBP4 >48.75 μg/mL (ORadj : 6.09 (1.3-28.57), p = 0.02) and GFAP <0.07 ng/mL (ORadj : 0.03 (0.003-0.31), p = 0.003) resulted in independent predictors of stroke subtype, improving discrimination by 29% (p < 0.0001). Both biomarkers might be useful as diagnostic biomarkers to differentiate ischemic stroke and ICH. A rapid differentiation of ischemic stroke from intracerebral hemorrhage is essential to provide the appropriate treatment. We describe the discovery and subsequent replications of RBP4 and its combination with circulating GFAP as plasmatic biomarkers for hyperacute stroke subtype differentiation. The combination of these biomarkers and others might aid to speed up the discrimination of both stroke subtypes improving the outcome of patients.

Bone marrow mononuclear cell (BM-MNC) intra-arterial transplantation improves recovery in experimental models of ischemic stroke through secretion of cytokines and growth factors (GFs), enhancing neoangiogenesis, and enhancing neuroplasticity. In this study, we tested whether BM-MNC transplantation in stroke patients induces changes in serum levels of cytokines and GFs. A phase I/II trial was conducted in middle cerebral artery (MCA) stroke patients with autologous intra-arterial BM-MNC transplantation between 5 and 9 days after stroke. Follow-up was done for up to 6 months. Eight cases and nine controls were included, and the serum levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), platelet-derived growth factor-BB (PDGF-BB), β nerve growth factor (β-NGF), and matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9) were measured before and 4, 8, and 90 days after transplantation. The correlation of these serum levels with dose of cells and clinical outcomes was studied. A total of 1.59 × 10 8 (±1.21 × 10 8 ) BM-MNCs were injected in cases; of them 3.38 × 10 6 (±2.33 × 10 6 ) were CD34 + cells. There was a positive correlation between total BM-MNCs injected and levels of GM-CSF and PDGF-BB at 90 days after transplantation ( r = 0.929, p = 0.001 and r = 0.714, p = 0.047, respectively), and a negative correlation between total CD34 + cells injected and MMP-2 levels at 4 days after transplantation ( r = −0.786, p = 0.036). Lower plasma levels of MMP-2 at 4 days and higher levels of PDGF-BB at 90 days were associated with better functional outcomes during follow-up ( p = 0.019 and p = 0.037, respectively). When administered intra-arterially in subacute MCA stroke patients, BM-MNCs seem to induce changes in serum levels of GM-CSF, PDGF-BB, and MMP-2, even 3 months after transplantation, which could be associated with better functional outcomes. This manuscript is published as part of the International Association of Neurorestoratology (IANR) special issue of Cell Transplantation.

Acute ischemic stroke is a complex disease with huge interindividual evolution variability that makes challenging the prediction of an adverse outcome. Our aim was to study the association of bloodstream signatures to early neurological outcome after stroke, by combining a subpooling of samples strategy with protein array discovery approach. Plasma samples from 36 acute stroke patients (< 4.5h from onset) were equally pooled within outcome groups: worsening, stability, and improvement (n = 3 pools of four patients each, for each outcome group). These nine pools were screened using a 177 antibodies library, and 35 proteins were found altered regarding outcome classification (p < 0.1). Processes of inflammation, immune response, coagulation, and apoptosis were regulated by these proteins. Ten representative candidates, mainly cytokines and chemokines, were assayed for replication in individual baseline plasma samples from 80 new stroke patients: beta-defensin2, MIP-3b, plasminogen activator inhibitor 1 active, beta-cell-attracting chemokine 1, Exodus-2, interleukin-4 receptor (IL-4R), IL-12p40, leukemia inhibitor factor, MIP-1b, and tumor necrosis factor-related weak inducer of apoptosis. Multivariate logistic regression analysis showed beta-defensin 2 (ORadj 4.87 [1.13-20.91] p = 0.033) and IL-4R (ORadj 3.52 [1.03-12.08] p = 0.045) as independent predictors of worsening at 24h after adjustment by clinical variables. Both biomarkers improve the prediction by 19% as compared to clinical information, suggesting a potential role for risk stratification in acute thrombolyzed stroke patients.

Endothelial progenitor cells (EPCs) represent a promising approach for cell-based therapies to induce tissue repair; however, their effective delivery into the brain has remained a challenge. We loaded EPCs with superparamagnetic iron oxide nanoparticles (SPIONs), assessed their angiogenic potential and evaluated their guidance to the brain using an external magnet. SPIONs were stored in the cytoplasm within endosomes/lysosomes as observed by transmission electronmicroscopy (TEM) and could be visualized as hypointense signals by magnetic resonance imaging (MRI) T-2-weighted images. In vitro SPION-loaded EPCs were fully functional, forming vessel-like structures in Matrigel (R), and displayed enhanced migration and secretion of growth factors (VEGF and FGF), which was associated with a moderate increase in reactive oxygen species production. Furthermore, in vivo MRI of treated mice showed accumulated hypointense signals consistent with SPION-loaded EPCs engraftment. Thus, we demonstrate that loading EPCs with SPIONs represents a safe and effective strategy for precise cell guidance into specific brain areas.From the Clinical Editor: This study investigates the potential role of endothelial progenitor cells in neuro-repair strategies of the central nervous system using SPION-loaded EPCs and magnetic guidance to the target organ. The authors demonstrate ex vivo cellular viability and maintained function following SPION load as well as successful guidance of the EPCs to the target site via MR imaging in a murine model. (C) 2014 Elsevier Inc. All rights reserved.

The enhancement of endogenous angiogenesis after stroke will be critical in neurorepair therapies where endothelial progenitor cells ( EPC s) might be key players. Our aim was to determine the influence of cerebral ischaemia and the role of matrix metalloproteinase‐9 ( MMP ‐9) on the angiogenic function of EPC s. Permanent focal cerebral ischaemia was induced by middle cerebral artery ( MCA ) occlusion in MMP ‐9/knockout (MMP‐9/KO) and wild‐type ( WT ) mice. EPC s were obtained for cell counting after ischaemia (6 and 24 hrs) and in control animals. Matrigel ™ assays and time‐lapse imaging were conducted to monitor angiogenic function of WT and MMP 9‐deficient EPC s or after treatment with MMP ‐9 inhibitors. Focal cerebral ischaemia increased the number of early EPC s, while MMP ‐9 deficiency decreased their number in non‐ischaemic mice and delayed their release after ischaemia. Late outgrowth endothelial cells ( OEC s) from ischaemic mice shaped more vessel structures than controls, while MMP ‐9 deficiency reduced the angiogenic abilities of OEC s to form vascular networks, in vitro . Treatment with the MMP inhibitor GM6001 and the specific MMP ‐9 inhibitor I also decreased the number of vessel structures shaped by both human and mouse WT OEC s, while exogenous MMP ‐9 could not revert the impaired angiogenic function in MMP ‐9/ KO OEC s. Finally, time‐lapse imaging showed that the extension of vascular networks was influenced by cerebral ischaemia and MMP ‐9 deficiency early during the vascular network formation followed by a dynamic vessel remodelling. We conclude that focal cerebral ischaemia triggers the angiogenic responses of EPC s, while MMP ‐9 plays a key role in the formation of vascular networks by EPC s.

Despite being ischemic stroke a leading cause of death and functional disability, there are no other accurate tools to predict outcome of patients beyond clinical variables such as age and stroke severity. In this scenario, defining protein changes associated with acute ischemic brain damage might help to identify new biomarker candidates for stroke prognosis. By means of mass spectrometry-based proteomics, we identified 51 proteins which levels were altered in the infarcted area of the human brain after stroke. Among 8 selected protein candidates, circulating levels of gelsolin, dihydropyrimidinase-related protein 2 and cystatin A were independent predictors of poor outcome. Logistic regression models including these innovative biomarkers significantly improved the predictive value with respect to the only use of clinical variables in both discrimination and reclassification analyses. Our results indicate that early blood determination of these three biomarkers might predict outcome of patients and might help in decision-making processes related to ischemic stroke management.Biological significanceCirculating levels of gelsolin, dihydopyrimidinase-related protein 2 and cystatin A, proteins found altered in human brain after cerebral ischemia, demonstrate potential usefulness as biomarkers for long-term stroke prognosis. (C) 2013 Elsevier B.V. All rights reserved.

BACKGROUND AND PURPOSE:Bone marrow mononuclear cell (BM-MNC) intra-arterial transplantation improves recovery in experimental models of ischemic stroke. We aimed to assess the safety, feasibility, and biological effects of autologous BM-MNC transplantation in patients with stroke.METHODS:A single-blind (outcomes assessor) controlled Phase I/II trial was conducted in patients with middle cerebral artery stroke. Autologous BM-MNCs were injected intra-arterially between 5 and 9 days after stroke. Follow-up was done for up to 6 months and blood samples were collected for biological markers. The primary outcome was safety and feasibility of the procedure. The secondary outcome was improvement in neurological function.RESULTS:Ten cases (BM-MNC-treated) and 10 control subjects (BM-MNC-nontreated) were consecutively included. Mean National Institutes of Health Stroke Scale before the procedure was 15.6. Mean BM-MNCs injected were 1.59×10(8). There was no death, stroke recurrence, or tumor formation during follow-up, although 2 cases had an isolate partial seizure at 3 months. After transplantation, higher plasma levels of beta nerve growth factor (β-nerve growth factor) were found compared with control subjects (P=0.02). There were no significant differences in neurological function at 180 days. A trend to positive correlation between number of CD34+ cells injected and Barthel Index was found (r=0.56, P=0.09).CONCLUSIONS:Intra-arterial BM-MNC transplantation in subacute ischemic stroke is feasible and seems to be safe. Larger randomized trials are needed to confirm the safety and elucidate the efficacy of BM-MNC transplantation.CLINICAL TRIAL REGISTRATION URL:www.clinicaltrials.gov. Unique identifier: NCT00761982.

Background: The induction of angiogenesis after stroke may enhance neurorestorative processes. Our aim was to examine the endogenous angiogenesis balance and their association with long-term clinical outcome in ischemic stroke patients.Methods: A total of 109 stroke subjects were included in the study. Firstly, plasma samples were obtained from control subjects (n = 26) and tPA-treated stroke patients (n = 29) at baseline (within 3 h of symptoms onset), 1, 2, 12, 24 h after tPA treatment, at discharge and 3 months after the ischemic event. Angiogenic promoters (PDGF-AA, PDGF-BB, HGF, FGF, KGF, HB-EGF, TPO, VEGF, VEGFR-1, VEGFR-2 and SDF-1 alpha) and inhibitors (endostatin, angiostatin, thrombospondin-1 and thrombospondin-2) were analyzed by Searchlight (R) technology or ELISA. Additionally, baseline and 24 h endostatin plasma level was determined in anewset of stroke patients (n = 80). Clinical parameters (NIHSS, mRS, mortality and hemorrhagic transformation events) were assessed to evaluate outcome.Results: Baseline PDGF-BB, endostatin and thrombospondin-2 levels were higher in stroke patients than in controls (p < 0.05). A pro-angiogenic balance was associated with lower NIHSS scores and less intracranial hemorrhagic complications. Interestingly, a high baseline endostatin level was associated to long-term functional dependency (mRS > 2; p = 0.004). Finally, a baseline endostatin cut-off point of 184 ng/mL was an independent predictor of functional dependency at three months in the multiple logistic regression with an odds ratio of 8.9 (95% CI: 2.7-28.8; p = 0.0002).Conclusions: Our results indicate that an early pro-angiogenic balance is associated with mild short-term neurological deficit, while an acute anti-angiogenesis status determined by high endostatin plasma level predicts a worse long-term functional outcome. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Dipyridamole (DP) is a platelet inhibitor with known antithrombotic benefits in stroke prevention. In addition to its anti-aggregant properties, recent studies have reported that DP promotes anti-inflammatory, anti-oxidative and neuroprotective effects. We aimed to test whether post-treatment with DP may exert protection after ischemic cerebral injury in the rat. For this purpose, rats were subjected to 120 min or 90 min of middle cerebral artery occlusion (MCAO) followed by 24 or 48 h of reperfusion, respectively. Either DP (100 mg/kg) or vehicle was administered i.v. at the onset of reperfusion; rats subjected to 90 min MCAO also received additional doses of DP orally (60 mg/kg) at 24 and 36 h after ischemia. Matrix metalloproteinases, extravasated hemoglobin content and IL-6, MIP-1α and MCP-1 cytokine level were examined in brain tissue by zymography, western blot and multiple ELISA, respectively. DP post-treatment led to a neurological improvement in both models (p < 0.05) and a significant reduction in the infarct volume of rats subjected to 90 min of ischemia, as compared to vehicle group (7.9% vs. 24.4%, p = 0.03). This neuroprotection was accompanied by a modest increase in expression of MMP-9 pro-form and a significant attenuation of MIP-1α levels in the infarcted hemisphere. These results provide support for the development of novel therapies based on DP for acute treatment of stroke. In selected animals, intravenous administration of high dose DP induced an adverse hypotensive effect leading to rapid death. Thus, alternative ways of acute administration must be examined in order to avoid this unfavorable effect.

Background: Vascular adhesion protein-1 (VAP-1) is a cell surface and circulating enzyme that belongs to the semicarbazide-sensitive amine oxidase (SSAO) family, which oxidatively deaminates primary amines and is implicated in leukocyte extravasation. Our aim was to investigate the alteration of soluble VAP-1/SSAO activity in plasma samples after acute intracerebral hemorrhage (ICH) and its presence in human ICH brain tissue. Methods: VAP-1/SSAO activity was determined in plasma of 66 ICH patients and 58 healthy controls. In addition, we assessed the expression of VAP-1/SSAO in postmortem brain tissue from hemorrhagic stroke patients by Western blot and immunohistochemistry. Results: We observed significantly higher levels of plasma VAP-1/SSAO activity in patients with ICH compared to matched elderly controls (p = 0.001). Plasma VAP-1/SSAO activity <2.7 pmol/min·mg and baseline ICH volume <17 ml were independent predictors of neurological improvement after 48 h (OR 6.8, 95% CI 1.14–41.67, p = 0.035, and OR 10.64, 95% CI 1.1–100, p = 0.041, respectively), after adjustment for baseline stroke severity. We also found that membrane-bound VAP-1/SSAO levels were lower in the perihematoma region than in the corresponding contralateral brain areas of patients deceased due to ICH (p = 0.024). Conclusions: Our data demonstrate that plasma VAP-1/SSAO activity is increased in ICH and predicts neurological outcome, suggesting a possible contribution of the soluble protein in secondary brain damage. Furthermore, anti-VAP-1/SSAO strategies might be a promising approach to prevent neurological worsening following ICH.

Background and purpose Circulating lipoprotein-associated phospholipase A2 (Lp-PLA2) has emerged as a novel biomarker for cardiovascular diseases. Our aim was to determine Lp-PLA2 mass and activity in a selected cohort of first-ever transient ischemic attack (TIA) or ischemic stroke patients with intracranial atherosclerotic disease (ICAD) and to investigate its relationship with the presence of classical vascular risk factors, response to secondary prevention treatments and risk of recurrent vascular events. Methods Lp-PLA2 mass and activity were measured 3 months after TIA or stroke by means of the PLAC test and CAM-assay (diaDexus, Inc.) respectively in 75 patients. Classic vascular risk factors, preventive treatments and clinical characteristics at the time of the index event were recorded. Follow-up transcranial Doppler ultrasonography (TCD) was performed and the presence of a new vascular event was assessed every 6 months. Results Several preventive treatments (statins and clopidogrel) were significantly associated with lower Lp-PLA2 mass and activity. During follow-up (median time 23 months), eighteen patients (24%) suffered a new vascular event. Baseline factors associated with new vascular events were: history of coronary artery disease, number of intracranial stenoses detected by TCD and also Lp-PLA2 activity, which was the only independent predictor for new vascular events (hazard ratio 2.89; 95% CI 1.029 to 8.096; p = 0.044) after multivariate analysis (Cox regression). Conclusions Lp-PLA2 activity might be a useful tool to identify intracranial large-artery occlusive disease patients at higher risk of suffering new vascular events.

Cerebral amyloid angiopathy (CAA) is one of the main causes of intracerebral hemorrhage (ICH) in the elderly. Matrix metalloproteinases (MMPs) have been implicated in blood–brain barrier disruption and ICH pathogenesis. In this study, we determined the levels MMP‐2 and MMP‐9 in plasma and their brain expression in CAA‐associated hemorrhagic stroke. Although MMP‐2 and MMP‐9 plasma levels did not differ among patients and controls, their brain expression was increased in perihematoma areas of CAA‐related hemorrhagic strokes compared with contralateral areas and nonhemorrhagic brains. In addition, MMP‐2 reactivity was found in β‐amyloid (Aβ)‐damaged vessels located far from the acute ICH and in chronic microbleeds. MMP‐2 expression was associated to endothelial cells, histiocytes and reactive astrocytes, whereas MMP‐9 expression was restricted to inflammatory cells. In summary, MMP‐2 expression within and around Aβ‐compromised vessels might contribute to the vasculature fatal fate, triggering an eventual bleeding.