The use of plasma biomarkers is becoming increasingly popular in several fields of medicine. In fact, decision-making processes using biomarkers is widely accepted in medical situations such as initiating lipid lowering therapies (LDL), diagnosing acute myocardial infarction (troponins), and ruling out pulmonary embolism suspicions (D-dimer), among others.

Therefore we really believe that biochemical markers of stroke, will really open “a window to the brain…”. In fact, in this research line we aim to answer relevant clinical questions through the use of biomarkers.

Our main Objectives using mainly plasma proteins are:

1.- To predict stroke risk

2.- To make stroke diagnosis

3.- To differentiate stroke subtypes

4.- To establish evolution and prognosis

5.- To use Biomarkers as treatment end-points

Some of our findings might have therapeutic implications since biological markers described by the group such as MMP-9 are well associated with Blood Brain Barrier disruption. In this direction, we have described MMP-9 predicting haemorrhagic complications among stroke patients receiving thrombolytic treatment. These approaches might contribute to increase safety of reperfusion treatments. The impact of this research line is clear, since articles like this (Circulation 2003) have been cited more than 180 times since its publication.

The study of these molecules will also have diagnostic implications because we have proposed the biochemical diagnostic of stroke by means of the identification of a biomarkers panel that distinguish between a stroke and other stroke-mimicking conditions. This might contribute to refer only to real stroke patients to the stroke centres, saving huge resources to the system.

These two examples, identification of biomarkers to predict tPA related bleedings and a stroke diagnostic test are examples of translational research in which the Neurovascular Research Lab is filing patents to be licensed to Biotech companies able to develop diagnostic kits in which our biomarkers might be placed and used in the clinical practice. That might close the circle of applied research.

Reviews of our group in which you may find detailed info about those biomarkers:

Foerch C, Montaner J, Furie KL, Ning MM, Lo EH.

      Searching for oracles? Blood biomarkers in acute stroke

      Neurology. 2009;73(5):393-9.

Montaner J.

      Blood biomarkers to guide stroke thrombolysis.

      Front Biosci (Elite Ed). 2009 Jun 1;1:200-208.

García-Berrocoso T, Fernández-Cadenas I, Delgado P, Rosell A, Montaner J.

      Blood Biomarkers to Identify Stroke Etiologies.

      Therapy 2010 (in press).

Projects

We are involved in several projects with public or private funding supporting our research on stroke biomarkers. One of such exciting projects is FIS PI 08/361 “Identificación y uso de biomarcadores pronósticos en el ictus isquémico”, aiming to identify biomarkers that predict main causes of stroke worsening (Infarct growth, cardiac complications, hemorrhagic transformation, infections, recurrence or new vascular events) to guide Stroke Unit allocation and stay of our patients.

International Collaborations

Discovery and validation of good candidate biomarkers, requires multidisciplinary approaches and replication in well designed multicentric and international studies. For that purpose we are actively collaborating with well known stroke research leading groups:

- Eng Lo, Xiaoing Wang, Ming Ming Ning (Boston, USA).

- Jean Charles Sanchez, Natacha Turck (Geneva, Switzerland).

- Denis Vivien, Carine Ali, Eduardo Anglés-Cano (Caen, France).

Research Tools and Techniques

We are using a combination of discovery techniques and biological human and animal samples to identify new stroke-related biomarkers:

One example is the “protein arrays”. In our laboratory, we have been using these new technologies and we actually use the SearchLight Assays (Aushon Biosystems) that allows us to study more than 170 human proteins and also many candidates from different animal species. We offer access to our technology to other groups. For more information, please refer to http://www.lin-bcn.com/

Also those biomarkers may be specifically tested not only as circulating markers in plasma or serum but in the components of interest of the Neurovascular Unit, that we may dissect by using the LCM Leica LMD6000 microscope, for Laser Capture microdissection. An example in which combine both thecniques may be found at:

Cuadrado E, Rosell A, Penalba A, Slevin M, Alvarez-Sabin J, Ortega-Aznar A,  Montaner J. Vascular MMP-9/TIMP-2 and neuronal MMP-10 up-regulation in human brain after stroke: a combined Laser Microdissection and Protein Array  Study. Journal of Proteome Research 2009;8(6):3191-3197.

Biobanks

All these results have been possible since the development of a “blood library” including more than 2000 stroke samples and a “brain library” that allowed us to describe for the first time the “human stroke proteome” with the outstanding collaboration of so many patients, relatives and clinicians of the Stroke Unit.

BACKGROUND

The only approved stroke treatment so far is the acute thrombolytic therapy with recombinant tissue plasminogen activator (rt-PA) when administered within 3-4.5 hours after symptoms onset. However, a reduced number of patients (5-8%) profit by this treatment, primarily because of the narrow therapeutic time-window and the risk of brain bleedings beyond thromrbolytic therapy to achieve the recanalization of the occluded artery. Moreover, the inflammatory response that accompanied necrotic brain injury contributes to aggravate acutely the progression of ischemic pathology. Inflammatory and brain damaged cells release a variety of cytotoxic agents including cytokines, MMPs and ROS which induce more cell damage as well as disruption of BBB and brain edema.

Thereby, it would be desirable to improve the efficacy and safety for thrombolytic therapy of stroke using combined anti-inflammatory strategies that may ameliorate the ischemic injury and means the best therapy translated at the clinical level.

Our research, conducted on experimental models of cerebral ischemia, is focused on the development of neuroprotective strategies aimed to salvage ischemic brain tissue by means complementary to reperfusion. Our goal is to find out a multimodal treatment that combines the administration of tPA together with other co-agents (as simvastatin and/or anti-aggregants with anti-inflammatory and neuro-protective properties) in attempt to obtain the most therapeutic benefice in the acute phase of ischemic stroke.

EXPERIMENTAL MODELS AND TECHNIQUES

Ischemic stroke has a complex pathophysiology involving the interplay of many different cells and tissues. Animal models of ischemic stroke are procedures inducing cerebral ischemia, which mimicked satisfactorily this cerebrovascular disease. Therefore, research stroke conducted on animal models has been shown essential for treatment approach of ischemic stroke.

Intra-arterial Suture Occlusion Model of Focal Cerebral Ischemia

Infarction in the territory of the middle cerebral artery (MCA) is induced by extracranial vascular occlusion on rat. A heat-blunted 4/0 nylon-monofilament is used to block the origin of MCA during 90 or 120 min and then, the monofilament is removed and reperfusion is allowed during the followed 24, 48 or 72 h.

This model mimics a clinical situation where recanalization of the occluded artery takes place, allowing test neuroprotectants that could be potentially co-administered with reperfusion agents like tPA.

Embolic Model of Focal Cerebral Ischemia

The stroke animal model by clot embolism has a large scientific interest in the clinical field because the high incidence of human thrombotic stroke and the use of reperfusion therapy with tPA, which can be assay on this model. Thromboembolic occlusion at the proximal level of MCA is achieved by injection of a rich fibrin clot through the internal carotid artery in the rat. Clots are previously performed using arterial blood from a donor rat. Thrombolytic therapy with tPA (9 mg/Kg) is administered by tail vein infusion (2 mg/mL;75 ?l/min) at different times points (2, 3, 4 hours) after occlusion.

Relative Cerebral Blood Flow (rCBF) is measured in the cortex supplied by the MCA by continuous Laser Doppler flowmetry (LDF) to ensure the successful of the artery occlusion or reperfusion in these models.  To examine the grade of infarction on rats, analysis of the infarct volume is evaluated using 2,3,5-triphenyltetrazolium chloride (TTC) staining and neurological score is assayed with a 9-point neurological at baseline (1-2 h) and each 24 h after occlusion.

NMR Imaging

Bruker-BIOSPEC 70/30 USR, 7 T Preclinical MRI System is used for the neuroimaging studies. Neuroimaging studies are conducted in vivo to valuate the cytotoxic edema and ischemic lesion (DWI, ADC map and T2WI) in the acute phase. Specific sequences are performed to assess Brain-Blood Barrier disruption (DCE) and occurrences of intracranial hemorrhages (T2*WI). Angiography is performed to document MCA occlusion or recanalization.

New therapies beyond the hyperacute phase of stroke are needed to be able to treat much more patients in delayed phases of this devastating disease. The idea that neurovascular plasticity contributes to stroke recovery can be a powerful concept for stroke therapy. Obviously, the therapeutic time window for interventions based on promoting recovery would be much larger than those for targeting acute stroke.  In this context, long-term neuroreparative therapies will  have to target the two essential phenomena to achieve brain neurorecovery after stroke: to restore the cerebral blood flow and to promote Neuroregeneration.

To achieve these major goals, both angiogenesis and neurogenesis need to be enhanced in the ischemic brain.

Classically, the formation of new blood vessels was thought to be mediated exclusively by embryogenic vasculogenesis followed by the sprouting of endothelial cells from preexisting vessels during angiogenesis. In the last decade, this standard dogma was overturned with the identification of the existence of circulating bone marrow-derived endothelial progenitor cells (EPCs). These cells are capable of differentiating, ex vivo, into endothelial-phenotyped cells, and now comprise a new model for endothelial generation and vessel repair (Asahara et al., 1997). These cells comprise a potential cell-based and growth-factor source of an alternate approach to enhance angio-neurogenic responses. 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 thorough specific growth factors.

Angiogenesis and neurogenesis occur endogenously after stroke. Our goal is to study these two complex phenomena both in experimental and human studies to finally potentiate them correctly to improve brain function and neurorecovery 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 presents very low mortality rates allowing long-term studies. Besides, the infarct is restricted to the cortex with clear boundary areas with normal cerebral blood flow and never affects neuroblast-rich areas such as the subventricular zone (then, both angiogenesis and neurogenesis can occur).

Functional outcome is assessed by the cylinder and corner tests which have been reported to be appropriate test for this type of cortical infarcts. Besides, histology and immunohistochemistry studies are conducted to evaluate brain repair and angio-neurogenic processes.

Endothelial Progenitor Cell Cultures:

EPCs are obtained from the Mononuclear cell fraction of human blood and from mouse spleen. MNCs are cultured in fibronectin-coated plates with complete cell culture medium EGM-2MV.

Both in human and murine cell cultures yield an early EPC population (also called Circulating Angiogenic Cells) obtained at day 4-7 after plating and late outgrowth EPC colonies (also called Endothelial Colony Forming Cells) appear from day 10 as colonies with high proliferation capacity and tubulogenic capacity.

In vitro Oxygen-Glucose Deprivation: endothelial cells and Endothelial progenitor cells are challenged to a transitory Oxygen and Glucose deprivation to study their angio-vasculogenic responses to ischemia and to test how potential treatments that 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 assays or cell adhesion to a mature monolayer of endothelial cells. Our studies focus on the angio-vasculogenic responses of both Endothelial Progenitor Cells and mature endothelial cells such as the human cell line of microvascular endothelial cells (hCMEC/D3).

NMR Imaging: Bruker-BIOSPEC 70/30 USR, 7 T Preclinical MRI System is used for the neuroimaging studies. Neuroimaging studies are conducted in vivo to follow-up the ischemic lesion. Specific sequences are performed to assess axonal degeneration/regeneration and changes in cerebral blood flow and angiogenesis.

Current project: 

Involment of proteolytic systems in the progression of Cerebral Amyloid Angiopathy

Cerebral amyloid angiopathy (CAA) is produced by the accumulation of ß-amyloid protein within the meningeal and brain vessels. It is the second leading cause of cerebral hemorrhages. However, nowadays, factors related to brain bleedings following amyloid deposition are largely unknown. The understanding of the mollecular mechanisms that lead to cerebral hemorrhage may be the basis for future treatments.

Previous evidences of our group have shown that Matrix Metal•loproteinases (MMPs) are related to brain bleeding. Now, we aim to investigate the relationship between these proteolytic systems and the appearance of intracraneal hemorrages in CAA.

Our study includes:

1) The identification of both tissue and plasma biomarkers for the diagnosis and prognosis of CAA-related hemorrhages.

2) The search of the genetic markers related to proteolytic systems that could determine the risk of suffering a recurrence in CAA.

We are studying a cohort of probable CAA patients that have been recruited in Hospital Vall d’Hebron in collaboration with the Stroke Project of the Cerebrovascular Diseases Study Group (Spanish Society of Neurology).

3) The study of MMPs role in ß-amyloid stimulated vascular cells in vitro.

Cultured cells of the neurovascular unit are challenged with different ß-amyloid peptides and the implication of MMPs in ß-amyloid cleavage and cell toxicity are studied using cellular and molecular biology methodology.

For this purpose, we use the human cerebral endothelial cell line hCMEC/D3, primary cultures of human leptomeningeal smooth muscle cells and rat/mouse glial and neuronal cultures.

Thioflavin-S staining of fibillar ß-amyloid within brain vessels of CAA patient’s sections.

Related bibliography:

1)  Hernández-Guillamon M, Delgado P, Ortega L, Pares M, Rosell A, García-Bonilla  L, Fernández-Cadenas I, Borrell-Pagès M, Boada M, Montaner J. Neuronal TIMP-1 release accompanies astrocytic MMP-9 secretion and enhances astrocyte proliferation induced by beta-amyloid 25-35 fragment. J Neurosci Res. 2009 Jul;87(9):2115-25.

2) Rosell A, Ortega-Aznar A, Alvarez-Sabín J, Fernández-Cadenas I, Ribó M, Molina CA, Lo EH, Montaner J. Increased brain expression of matrix metalloproteinase-9 after ischemic and hemorrhagic human stroke. Stroke. 2006 Jun;37(6):1399-406.

3)  Alvarez-Sabín J, Delgado P, Abilleira S, Molina CA, Arenillas J, Ribó M, Santamarina E, Quintana M, Monasterio J, Montaner J. Temporal profile of matrix metalloproteinases and their inhibitors after spontaneous intracerebral hemorrhage: relationship to clinical and radiological outcome. Stroke. 2004 Jun;35(6):1316-22.

4) Montaner J, Molina CA, Monasterio J, Abilleira S, Arenillas JF, Ribó M, Quintana M, Alvarez-Sabín J. Matrix metalloproteinase-9 pretreatment level predicts intracranial hemorrhagic complications after thrombolysis in human stroke. Circulation. 2003 Feb 4;107(4):598-603.

Collaborations:

Mercè Boada

Fundació ACE, Barcelona, Spain.

www.fundacioace.com/

Jorge Ghiso and Agueda Rostagno

Pathology Dept. Langone Medical Center. NYU

New York, US.

Ignacio Romero

Life Science Dept. Open University.

Milton Keynes, UK.