New therapies beyond the hyperacute phase of stroke would be needed to permit the treatment of much more patients in later phases of this devastating disease. The hypothesis that neurovascular plasticity contributes to stroke recovery could be a powerful new concept to search for new stroke therapies since the therapeutic time window for interventions based on promoting recovery would be much larger than for those targeting the acute stroke only. Therefore, long-term neuroreparative therapies would have to target the two essential phenomena that allow brain neurorecovery after a stroke: restore the cerebral blood flow and promote neuroregeneration. To achieve these major goals, both spontaneous angiogenesis and neurogenesis need to be enhanced in the ischemic brain.
The formation of new blood vessels was usually thought to be mediated exclusively by embryogenic vasculogenesis, followed by the sprouting of endothelial cells from preexisting vessels during angiogenesis. However, during the last decade, this standard dogma was overturned with the discovery of circulating bone marrow-derived endothelial progenitor cells (EPCs). These cells are capable of differentiating ex vivo into endothelial-phenotyped cells, and thus represent a new model for endothelial generation and vessel repair (Asahara et al., 1997). These cells constitute a potential source of growth-factors, permitting then an alternate approach to enhance the angio-neurogenic response. In fact, newborn neurons (neurogenesis) and new vascular components (angiogenesis) form a microenvironment that has been termed the neurovascular niche [Ohab et al., 2006], were angiogenesis and neurogenesis are linked throughout specific growth factors. In this regard EPC offer double therapeutic potential since they can incorporate into new vessels as mature endothelial cells but are also a source of growth factors that regulate the function of brain resident cells such as endothelial cells, neuroblasts, mature neurons or oligodendrocyte precursors. Cell-free therapies based on the factors secreted by these cells are now being considered beyond the traditional cell-based therapies (Di Santo et al. 2009 and Horie et al. 2011).
The mechanisms that regulate the therapeutic potential of angiogenic factors and EPCs in stroke patients are now being investigated. Our goal is to study both angiogenesis and neurogenesis in experimental and human studies in order to identify how to potentiate them correctly and to how to improve brain function and neurorecovery after stroke from a translational prespective.
1) Experimental studies in animal models of cerebral ischemia to develop pro angiogenic therapies (EPC-based) to obtain maximal neuroreparative benefits following stroke.
2) Role of matrix proteinases in angiogenesis-triggered neurorepair therapies.
3) Experimental studies in animal models to investigate mechanisms of neurovascular coupling between angiogenesis and neurogenesis after stroke.
4) Biotechnological platforms to improve the delivery of EPCs and growth factors.
5) Study of EPCs’ secretome.
6) Biomarkers of angiogenesis and repair after stroke.
· EXPERIMENTAL MODELS AND TECHNIQUES:
In vivo stroke models: Cerebral ischemia affecting the cortical territory of the Middle Cerebral Artery (MCA) is occluded at the level of the M1 portion (distal occlusion). This model has been chosen because it presents very low mortality rates allowing long-term studies. Moreover, the infarct is usually restricted to the cortex with clear boundary areas with normal cerebral blood flow, and never affects neuroblast-rich areas such as the subventricular zone. We are currently performing two models of distal occlusion in mice: permanent and transient (60 minutes) occlusion of the MCA fully described at Morancho et al. 2012. We follow the ARRIVE recommendations to conduct our animal stadies and report our results.
Endothelial Progenitor Cell Cultures: EPCs are obtained from the mononuclear cell (MNC) fraction of human blood and from mouse spleen. MNCs are cultured in fibronectin-coated plates with complete cell culture medium EGM-2MV as described at Rosell et al 2009 and Navarro-Sobrino et al. 2010. Both in humans and mice, cell cultures yield an early EPC population (also called Circulating Angiogenic Cells) obtained at day 5-7 after plating, and late outgrowth colonies (also called Endothelial Colony Forming Cells) which might appear from day 10 as colonies with high proliferation capacity, endothelial phenotype and tubulogenic abilities.
In vitro Oxygen-Glucose Deprivation: endothelial cells and endothelial progenitor cells are challenged to a transitory oxygen and/or glucose deprivation (INVIVO200, Ruskinn, Cultek) in order to study their angio-vasculogenic capacity to respond to ischemia and to evaluate how potential treatments could modify these responses
Angiogenesis-related techniques: angio-vasculogenic mechanisms are studied in a variety of in vitro assays including Matrigel® tubulogenesis, cell migration using trans-well or scratch assays. Our studies focus on the angio-vasculogenic responses of both EPCs and mature endothelial cells such as the human cell line of microvascular endothelial cells (hCMEC/D3).
NMR Imaging: The Bruker-BIOSPEC 70/30 USR, 7 T Preclinical MRI System is used for the neuroimaging studies (Universitat Autònoma de Barcelona). In vivo mouse studies are conducted to examine the ischemic lesion, brain damage evolution and cell tracking in cell-therapy-based studies. Specific sequences are performed to estimate axonal degeneration/regeneration, changes in cerebral blood flow and angiogenesis.
- Anna Rosell, PhD. Miguel Servet Position.
- Anna Morancho, Lab Technician.
- Fei-fei Ma, MSc. PhD Studen.
- Marina Gabriel, MSc, PhD Student
- Dr. Santiago Roure, Grup de Recerca en insuficiència Cardíaca i Regeneració. IGTP- Badalona
- Dr. Zenon Starcuk and Radovan Jirik. Institute of Scientic Instruments, Academy of Sciences of the Czech Republic
- Dra. Susana Rodríguez and Dra. Immaculada Bori. Unitat de Neurorehabilitació i Dany Cerebral- HUVH
- Anna Roig, Elisa Carenza and Anna Laromaine. Institut Ciències de Materials-CSIC, Cerdanyola del Vallès, Spain.
- Jari Koistinaho. A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland.
- José Páramo and Josune Orbe. CIMA, Clínica Universitaria de Navarra, Pamplona, Spain.
- Dennis Vivien and Carine Ali, INSERM UMR-S U919 Université Caen Basse-Normandie, GIP CYCERON, Caen, France.
- Eng H. Lo and Ken Arai. Neuroprotection Research Laboratory. Massachusetts General Hospital, Boston, MA, USA.
- Magnetic nanoparticles suspensions for cell tracking, imaging and therapeutics in ischemia treatments.
Elisa Carenza. Universitat Autònoma de Barcelona, 2014
- Neurorepair Treatments for Cerebral Ischemia using Endothelial Progenitor Cells (EPCs).
Anna Morancho Retana.Universitat Autònoma de Barcelona, 2014.
- Influence of Endothelial Progenitor Cells on spatial and temporal modulation of Angiogenesis and Vasculogenesis following human ischemic stroke.
Miriam Navarro Sobrino, Universitat Autònoma de Barcelona, 2010.
- Matrix Metalloproteinases in cerebral ischemia: cellular origin and tissue damage contribution.
Eloy Cuadrado Godia, Universitat Autònoma de Barcelona, 2009.
- Dual Role of Matrix Metalloproteinases in brain injury and neurorepair after cerebral ischemia Feifei Ma, Universitat Autònoma de Barcelona, 2015.
Ma F, Martínez-San Segundo P, Barceló V, Morancho A, Gabriel-Salazar M, Giralt
D, Montaner J, Rosell A. Matrix metalloproteinase-13 participates in
neuroprotection and neurorepair after cerebral ischemia in mice. Neurobiol Dis.
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that preserves survival pathways by preventing Kidins220/ARMS calpain processing
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remodeling after endothelial progenitor cell transplantation in MMP9-deficient
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Parés M, Fernández-Cadenas I, Giralt D, Carrera C, Ribó M, Vivien D, Ali C,
Rosell A, Montaner J. NURR1 involvement in recombinant tissue-type plasminogen
activator treatment complications after ischemic stroke. Stroke. 2015
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Cayuela A, Escudero I, de la Torre-Laviana FJ, Boada C, Rosell A, Mayol A,
Jimenez MD, Gil-Peralta A, Gonzalez A. 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. Cell Transplant. 2014;23 Suppl
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Campos-Martorell M; Ma Ángeles Montero, Hernández-Guillamón M, Rosell A, Gomis
J, Salat D, García-Bonilla L, Montaner J. Rat middle cerebral artery occlusion is
not a suitable model for the study of stroke-induced spontaneous infections. PLoS
One. 2014 Jun 12;9(6):e99169. doi: 10.1371/journal.pone.0099169. eCollection
2014. PubMed PMID: 24922549; PubMed Central PMCID: PMC4055651.
Carenza E, Barceló V, Morancho A, Montaner J, Rosell A, Roig A. Rapid
synthesis of water-dispersible superparamagnetic iron oxide nanoparticles by a
microwave-assisted route for safe labeling of endothelial progenitor cells. Acta
Biomater. 2014 Aug;10(8):3775-85. doi: 10.1016/j.actbio.2014.04.010. Epub 2014
Apr 20. PubMed PMID: 24755438.
Campos-Martorell M, Salvador N, Monge M, Canals F, García-Bonilla L,
Hernández-Guillamon M, Ayuso MI, Chacón P, Rosell A, Alcazar A, Montaner J. Brain
proteomics identifies potential simvastatin targets in acute phase of stroke in a
rat embolic model. J Neurochem. 2014 Jul;130(2):301-12. doi: 10.1111/jnc.12719.
Epub 2014 Apr 30. PubMed PMID: 24661059.
Llombart V, Dominguez C, Bustamante A, Rodriguez-Sureda V, Martín-Gallán P,
Vilches A, García-Berrocoso T, Penalba A, Hernández-Guillamon M, Rubiera M, Ribó
M, Eschenfelder C, Giralt D, Molina CA, Alvarez-Sabín J, Rosell A, Montaner J.
Fluorescent molecular peroxidation products: a prognostic biomarker of early
neurologic deterioration after thrombolysis. Stroke. 2014 Feb;45(2):432-7. doi:
10.1161/STROKEAHA.113.003431. Epub 2013 Dec 12. PubMed PMID: 24335228.
Rosell A, Agin V, Rahman M, Morancho A, Ali C, Koistinaho J, Wang X, Vivien
D, Schwaninger M, Montaner J. Distal occlusion of the middle cerebral artery in
mice: are we ready to assess long-term functional outcome? Transl Stroke Res.
2013 Jun;4(3):297-307. doi: 10.1007/s12975-012-0234-1. Epub 2013 Jan 9. Review.
PubMed PMID: 24323300.
Campos M, García-Bonilla L, Hernández-Guillamon M, Barceló V, Morancho A,
Quintana M, Rubiera M, Rosell A, Montaner J. Combining statins with tissue
plasminogen activator treatment after experimental and human stroke: a safety
study on hemorrhagic transformation. CNS Neurosci Ther. 2013 Nov;19(11):863-70.
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