Cristina Boada

Intra-Arterial Bone Marrow Mononuclear Cells in Ischemic Stroke A Pilot Clinical Trial

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 1.59×108. 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 (&bgr;-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.

A large screening of angiogenesis biomarkers and their association with neurological outcome after ischemic stroke.

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 3h of symptoms onset), 1, 2, 12, 24h 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-1α) and inhibitors (endostatin, angiostatin, thrombospondin-1 and thrombospondin-2) were analyzed by Searchlight(®) technology or ELISA. Additionally, baseline and 24h endostatin plasma level was determined in a new set 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.

MMP-2/MMP-9 plasma level and brain expression in cerebral amyloid angiopathy-associated hemorrhagic stroke.

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.

In vitro angiogenic performance and in vivo brain targeting of magnetized endothelial progenitor cells for neurorepair therapies

UNLABELLED 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 electron microscopy (TEM) and could be visualized as hypointense signals by magnetic resonance imaging (MRI) T2-weighted images. In vitro SPION-loaded EPCs were fully functional, forming vessel-like structures in Matrigel®, 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.

VAP-1/SSAO Plasma Activity and Brain Expression in Human Hemorrhagic Stroke

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.

Effects of acute post-treatment with dipyridamole in a rat model of focal cerebral ischemia

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 (100mg/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.

Intra-Arterial Bone Marrow Mononuclear Cell Transplantation Correlates with GM-CSF, PDGF-BB, and MMP-2 Serum Levels in Stroke Patients: Results from a Clinical Trial:

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 × 108 (±1.21 × 108) BM-MNCs were injected in cases; of them 3.38 × 106 (±2.33 × 106) 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.

Lipoprotein-associated phospholipase A2 testing usefulness among patients with symptomatic intracranial atherosclerotic disease

BACKGROUND AND PURPOSE Circulating lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) has emerged as a novel biomarker for cardiovascular diseases. Our aim was to determine Lp-PLA(2) 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-PLA(2) 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-PLA(2) 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-PLA(2) 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-PLA(2) activity might be a useful tool to identify intracranial large-artery occlusive disease patients at higher risk of suffering new vascular events.

Cerebral ischaemia and matrix metalloproteinase-9 modulate the angiogenic function of early and late outgrowth endothelial progenitor cells

The enhancement of endogenous angiogenesis after stroke will be critical in neurorepair therapies where endothelial progenitor cells (EPCs) 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 EPCs. Permanent focal cerebral ischaemia was induced by middle cerebral artery (MCA) occlusion in MMP‐9/knockout (MMP‐9/KO) and wild‐type (WT) mice. EPCs 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 MMP9‐deficient EPCs or after treatment with MMP‐9 inhibitors. Focal cerebral ischaemia increased the number of early EPCs, while MMP‐9 deficiency decreased their number in non‐ischaemic mice and delayed their release after ischaemia. Late outgrowth endothelial cells (OECs) from ischaemic mice shaped more vessel structures than controls, while MMP‐9 deficiency reduced the angiogenic abilities of OECs 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 OECs, while exogenous MMP‐9 could not revert the impaired angiogenic function in MMP‐9/KO OECs. 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 EPCs, while MMP‐9 plays a key role in the formation of vascular networks by EPCs.

Plasmatic retinol-binding protein 4 and glial fibrillary acidic protein as biomarkers to differentiate ischemic stroke and intracerebral hemorrhage

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.