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.
Esquiva G, Grayston A, Rosell A. Revascularization and endothelial progenitor
cells in stroke. Am J Physiol Cell Physiol. 2018 Nov 1;315(5):C664-C674. doi:
10.1152/ajpcell.00200.2018. Epub 2018 Aug 22. PubMed PMID: 30133323.
Gabriel-Salazar M, Morancho A, Rodriguez S, Buxó X, García-Rodríguez N, Colell
G, Fernandez A, Giralt D, Bustamante A, Montaner J, Rosell A. Importance of
Angiogenin and Endothelial Progenitor Cells After Rehabilitation Both in Ischemic
Stroke Patients and in a Mouse Model of Cerebral Ischemia. Front Neurol. 2018 Jun
29;9:508. doi: 10.3389/fneur.2018.00508. eCollection 2018. PubMed PMID: 30008694;
PubMed Central PMCID: PMC6034071.
Simats A, García-Berrocoso T, Ramiro L, Giralt D, Gill N, Penalba A,
Bustamante A, Rosell A, Montaner J. Characterization of the rat cerebrospinal
fluid proteome following acute cerebral ischemia using an aptamer-based proteomic
technology. Sci Rep. 2018 May 21;8(1):7899. doi: 10.1038/s41598-018-26237-3.
PubMed PMID: 29784938; PubMed Central PMCID: PMC5962600.
Hayakawa K, Chan SJ, Mandeville ET, Park JH, Bruzzese M, Montaner J, Arai K,
Rosell A, Lo EH. Protective Effects of Endothelial Progenitor Cell-Derived
Extracellular Mitochondria in Brain Endothelium. Stem Cells. 2018
Sep;36(9):1404-1410. doi: 10.1002/stem.2856. Epub 2018 Jul 15. PubMed PMID:
29781122; PubMed Central PMCID: PMC6407639.
Maki T, Morancho A, Martinez-San Segundo P, Hayakawa K, Takase H, Liang AC,
Gabriel-Salazar M, Medina-Gutiérrez E, Washida K, Montaner J, Lok J, Lo EH, Arai
K, Rosell A. Endothelial Progenitor Cell Secretome and Oligovascular Repair in a
Mouse Model of Prolonged Cerebral Hypoperfusion. Stroke. 2018
Apr;49(4):1003-1010. doi: 10.1161/STROKEAHA.117.019346. Epub 2018 Mar 6. PubMed
PMID: 29511131; PubMed Central PMCID: PMC5871569.
Simats A, García-Berrocoso T, Penalba A, Giralt D, Llovera G, Jiang Y, Ramiro
L, Bustamante A, Martinez-Saez E, Canals F, Wang X, Liesz A, Rosell A, Montaner
J. CCL23: a new CC chemokine involved in human brain damage. J Intern Med. 2018
May;283(5):461-475. doi: 10.1111/joim.12738. Epub 2018 Mar 5. PubMed PMID:
Percie du Sert N, Alfieri A, Allan SM, Carswell HV, Deuchar GA, Farr TD,
Flecknell P, Gallagher L, Gibson CL, Haley MJ, Macleod MR, McColl BW, McCabe C,
Morancho A, Moon LD, O’Neill MJ, Pérez de Puig I, Planas A, Ragan CI, Rosell A,
Roy LA, Ryder KO, Simats A, Sena ES, Sutherland BA, Tricklebank MD, Trueman RC,
Whitfield L, Wong R, Macrae IM. The IMPROVE Guidelines (Ischaemia Models:
Procedural Refinements Of in Vivo Experiments). J Cereb Blood Flow Metab. 2017
Nov;37(11):3488-3517. doi: 10.1177/0271678X17709185. Epub 2017 Aug 11. Review.
PubMed PMID: 28797196; PubMed Central PMCID: PMC5669349.
Roncal C, Martinez de Lizarrondo S, Salicio A, Chevilley A, Rodriguez JA,
Rosell A, Couraud PO, Weksler B, Montaner J, Vivien D, Páramo JA, Orbe J. New
thrombolytic strategy providing neuroprotection in experimental ischemic stroke:
MMP10 alone or in combination with tissue-type plasminogen activator. Cardiovasc
Res. 2017 Aug 1;113(10):1219-1229. doi: 10.1093/cvr/cvx069. PubMed PMID: