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2.
Neurobiol Dis ; 91: 236-46, 2016 07.
Article in English | MEDLINE | ID: mdl-27001146

ABSTRACT

New neuroreparative and neuroprotective therapies are being sought to treat stroke patients. One approach is the remodeling of extracellular matrix, which participates in both brain injury and neurovascular repair when matrix metalloproteinases (MMPs) are thought to be key players. Our aim was to investigate the role of MMP-13 (collagenase-3) in the acute (24h and 3days) and delayed (2weeks) phases of stroke. Permanent and transient cerebral ischemia models involving the cortex were induced in MMP-13 knock-out (KO) and wild-type (WT) mice. In the transient model, MMP-13 deficiency reduced the amount of TTC-stained infarct tissue, reduced hemorrhagic events and improved functional outcomes (p<0.01). At two weeks, normal neuroblast (DCX+) migration from the subventricular zone toward the peri-infarct area was observed. However, MMP-13 deficiency significantly reduced the number of newborn neuroblasts (DCX+/BrdU+) in the cortical peri-infarct area (p<0.01). This result occurred in parallel with aberrant cortical vascular remodeling: post-stroke peri-infarct vessel density increased in the WT mice (p<0.01) but this increase was blocked in the MMP-13 KO mice. Prior to these vascular alterations, the levels of pro-angiogenic factors, including G-CSF, VEGF-A and angiopoietin-2, were lower in the ischemic cortex of MMP-13 KO mice than in WT mice (p<0.05). In vitro, gene-silencing of MMP-13 in endothelial progenitor cells (EPCs) confirmed the reduced ability of these cells to build tubulogenic networks in Matrigel™ substrate. Together, our results indicate that MMP-13 is a central protease in infarct development and cortical remodeling during post-stroke neurorepair, which is critical for optimal angiogenic and neurogenic responses.


Subject(s)
Ischemic Attack, Transient/enzymology , Matrix Metalloproteinase 13/metabolism , Neuroprotection/physiology , Stroke/enzymology , Animals , Disease Models, Animal , Doublecortin Protein , Infarction, Middle Cerebral Artery/metabolism , Matrix Metalloproteinase 13/genetics , Mice, Knockout , Neurogenesis/physiology
3.
J Cereb Blood Flow Metab ; 35(10): 1547-51, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26219597

ABSTRACT

Endothelial progenitor cells (EPCs) are being investigated for advanced therapies, and matrix metalloproteinase 9 (MMP9) has an important role in stroke recovery. Our aim was to determine whether tissue MMP9 influences the EPC-induced angiogenesis after ischemia. Wild-type (WT) and MMP9-deficient mice (MMP9/KO) were subjected to cerebral ischemia and treated with vehicle or outgrowth EPCs. After 3 weeks, we observed an increase in the peri-infarct vessel density in WT animals but not in MMP9/KO mice; no differences were found in the vehicle-treated groups. Our data suggest that tissue MMP9 has a crucial role in EPC-induced vascular remodeling after stroke.


Subject(s)
Brain Ischemia/genetics , Cerebral Cortex/pathology , Cerebral Veins/physiology , Endothelial Progenitor Cells , Matrix Metalloproteinase 9/physiology , Stem Cell Transplantation/methods , Vascular Remodeling , Animals , Brain Ischemia/pathology , Brain Ischemia/therapy , Cerebral Infarction/pathology , Cerebral Infarction/physiopathology , Cerebral Veins/growth & development , DNA-Binding Proteins , Doublecortin Domain Proteins , Magnetic Resonance Imaging , Male , Matrix Metalloproteinase 9/deficiency , Matrix Metalloproteinase 9/genetics , Mice , Mice, Knockout , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/physiology , Neovascularization, Physiologic/genetics , Neovascularization, Physiologic/physiology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/physiology , Neuropeptides/genetics , Neuropeptides/physiology , Nuclear Proteins/genetics , Nuclear Proteins/physiology , Stroke/therapy
4.
Acta Biomater ; 10(8): 3775-85, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24755438

ABSTRACT

We synthesize highly crystalline citrate-coated iron oxide superparamagnetic nanoparticles that are stable and readily dispersible in water by an extremely fast microwave-assisted route and investigate the uptake of magnetic nanoparticles by endothelial cells. Nanoparticles form large aggregates when added to complete endothelial cell medium. The size of the aggregates was controlled by adjusting the ionic strength of the medium. The internalization of nanoparticles into endothelial cells was then investigated by transmission electron microscopy, magnetometry and chemical analysis, together with cell viability assays. Interestingly, a sevenfold more efficient uptake was found for systems with larger nanoparticle aggregates, which also showed significantly higher magnetic resonance imaging effectiveness without compromising cell viability and functionality. We are thus presenting an example of a straightforward microwave synthesis of citrate-coated iron oxide nanoparticles for safe endothelial progenitor cell labeling and good magnetic resonance cell imaging with potential application for magnetic cell guidance and in vivo cell tracking.


Subject(s)
Cell Tracking/methods , Dextrans/chemistry , Endothelial Progenitor Cells/chemistry , Endothelial Progenitor Cells/cytology , Magnetic Resonance Imaging/methods , Magnetite Nanoparticles/chemistry , Water/chemistry , Animals , Citric Acid/chemistry , Contrast Media/chemistry , Diffusion , Drug Compounding/methods , Endothelial Cells , Humans , Male , Mice , Mice, Inbred BALB C , Microwaves , Particle Size , Staining and Labeling/methods
5.
Nanomedicine ; 10(1): 225-34, 2014 Jan.
Article in English | MEDLINE | ID: mdl-23792330

ABSTRACT

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.


Subject(s)
Endothelial Cells/drug effects , Ferric Compounds/administration & dosage , Magnetite Nanoparticles/administration & dosage , Stem Cells/drug effects , Animals , Brain/diagnostic imaging , Brain/drug effects , Brain/metabolism , Cell Differentiation/drug effects , Endothelial Cells/metabolism , Magnetic Resonance Imaging , Magnetite Nanoparticles/chemistry , Mice , Microscopy, Electron, Transmission , Radiography , Reactive Oxygen Species/metabolism , Staining and Labeling , Stem Cells/metabolism , Vascular Endothelial Growth Factor A/biosynthesis
6.
CNS Neurosci Ther ; 19(11): 863-70, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24118905

ABSTRACT

AIMS: Statins may afford neuroprotection against ischemic injury, but it remains controversial whether combined treatment with tissue plasminogen activator (tPA) after stroke increases the risk of hemorrhagic transformation (HT), the major tPA-related complication. We evaluated the safety of combining statin with tPA administration during the acute phase of both experimental and human stroke. METHODS: The occurrence and severity of HT, infarct volume, and neurological outcome were evaluated in spontaneous hypertensive rats (SHR) subjected to embolic middle cerebral arterial occlusion (MCAO), which received vehicle or simvastatin (20 mg/kg), 15 min after ischemia and tPA (9 mg/kg) 3 h after ischemia. Additionally, HT rate was evaluated in stroke patients who were treated with tPA (0.9 mg/kg) within 3 h after symptom onset, considering whether or not were under statins treatment when the stroke occurred. RESULTS: In the experimental study, no differences in HT rates and severity were found between treatment groups, neither regarding mortality, neurological deficit, infarct volume, or metalloproteinases (MMPs) brain content. In the clinical study, HT rates and hemorrhage type were similar in stroke patients who were or not under statins treatment. CONCLUSION: This study consistently confirms that the use of statins does not increase HT rates and severity when is combined with tPA administration.


Subject(s)
Cerebral Hemorrhage/pathology , Hydroxymethylglutaryl-CoA Reductase Inhibitors/administration & dosage , Stroke/drug therapy , Stroke/pathology , Tissue Plasminogen Activator/administration & dosage , Aged , Aged, 80 and over , Animals , Cerebral Hemorrhage/chemically induced , Cohort Studies , Drug Therapy, Combination , Female , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/adverse effects , Male , Middle Aged , Pilot Projects , Prospective Studies , Random Allocation , Rats , Rats, Inbred SHR , Tissue Plasminogen Activator/adverse effects
7.
PLoS One ; 8(9): e73244, 2013.
Article in English | MEDLINE | ID: mdl-24023842

ABSTRACT

Cell therapy with endothelial progenitor cells (EPCs) has emerged as a promising strategy to regenerate the brain after stroke. Here, we aimed to investigate if treatment with EPCs or their secreted factors could potentiate angiogenesis and neurogenesis after permanent focal cerebral ischemia in a mouse model of ischemic stroke. BALB/C male mice were subjected to distal occlusion of the middle cerebral artery, and EPCs, cell-free conditioned media (CM) obtained from EPCs, or vehicle media were administered one day after ischemia. Magnetic resonance imaging (MRI) was performed at baseline to confirm that the lesions were similar between groups. Immunohistochemical and histological evaluation of the brain was performed to evaluate angio-neurogenesis and neurological outcome at two weeks. CM contained growth factors, such as VEGF, FGF-b and PDGF-bb. A significant increase in capillary density was noted in the peri-infarct areas of EPC- and CM-treated animals. Bielschowsky's staining revealed a significant increase in axonal rewiring in EPC-treated animals compared with shams, but not in CM-treated mice, in close proximity with DCX-positive migrating neuroblasts. At the functional level, post-ischemia forelimb strength was significantly improved in animals receiving EPCs or CM, but not in those receiving vehicle media. In conclusion, we demonstrate for the first time that the administration of EPC-secreted factors could become a safe and effective cell-free option to be considered in future therapeutic strategies for stroke.


Subject(s)
Brain Ischemia/therapy , Brain/pathology , Brain/physiopathology , Cell- and Tissue-Based Therapy/methods , Endothelial Cells/cytology , Stem Cells/cytology , Animals , Axons/pathology , Brain Ischemia/complications , Brain Ischemia/pathology , Brain Ischemia/physiopathology , Cell Movement , Culture Media, Conditioned/metabolism , Doublecortin Protein , Magnetic Resonance Imaging , Male , Mice , Mice, Inbred BALB C , Neovascularization, Physiologic , Stroke/complications , Treatment Outcome
8.
J Cell Mol Med ; 17(12): 1543-53, 2013 Dec.
Article in English | MEDLINE | ID: mdl-23945132

ABSTRACT

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.


Subject(s)
Brain Ischemia/enzymology , Brain Ischemia/physiopathology , Endothelial Cells/metabolism , Matrix Metalloproteinase 9/metabolism , Neovascularization, Physiologic , Stem Cells/metabolism , Animals , Brain Ischemia/pathology , Cell Count , Collagen/metabolism , Drug Combinations , Endothelial Cells/drug effects , Endothelial Cells/pathology , Humans , Immunophenotyping , Laminin/metabolism , Male , Matrix Metalloproteinase 9/deficiency , Matrix Metalloproteinase Inhibitors/pharmacology , Mice , Neovascularization, Physiologic/drug effects , Proteoglycans/metabolism , Stem Cells/drug effects , Stem Cells/pathology
9.
Neuropathol Appl Neurobiol ; 38(6): 617-27, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22289071

ABSTRACT

AIMS: Rodent experimental models are essential for in vivo study of stroke. Our aim was to develop a reproducible method of mouse transient focal cerebral ischaemia by distal artery compression. METHODS: The distal middle cerebral artery (dMCA) was occluded by compression with a blunted needle, and cerebral blood flow was monitored by laser Doppler flowmetry to ensure appropriate occlusion and reperfusion in Balb/c mice. The ischaemic lesion was evaluated 24 h after occlusion by TTC staining and immunolabelling (NeuN, CD31, GFAP and Iba-1) while the established permanent dMCA occlusion (dMCAO) model was used as a control. The corner test was performed to evaluate neurological behaviour. RESULTS: Laser Doppler flowmetry register showed a homogenous arterial occlusion among animals. Forty-five minutes of arterial occlusion did not lead brain infarction when evaluated by TTC staining 24 h after occlusion. Extending the cerebral ischaemia period to 60 min induced a cortically localized homogeneous brain infarct. No differences in infarct volume were detected between animals submitted to permanent or 60-min transient dMCAO (42.33 ± 9.88 mm³ and 37.63 ± 12.09 mm³ respectively). The ischaemic injury was confirmed by immunohistochemistry in the 60-min transient dMCAO model but not in the 45-min model. Neurological deficits assessed with the corner test were significant only during the first 48 h but not at long term. CONCLUSIONS: This work shows an easy-to-perform method for the induction of brain ischaemia and reperfusion to assess stroke repair and treatment screening, with cortically localized ischaemic cell damage, low mortality and neurological impairment in the acute phase.


Subject(s)
Brain/blood supply , Disease Models, Animal , Infarction, Middle Cerebral Artery/pathology , Middle Cerebral Artery/pathology , Stroke/pathology , Animals , Infarction, Middle Cerebral Artery/physiopathology , Mice , Middle Cerebral Artery/physiopathology , Stroke/physiopathology
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