Anna Penalba

A Matrix Metalloproteinase Protein Array Reveals a Strong Relation Between MMP-9 and MMP-13 With Diffusion-Weighted Image Lesion Increase in Human Stroke

Background and Purpose— Matrix metalloproteinases (MMPs) are involved in tissue destruction produced by the neuroinflammatory response that follows ischemic stroke. In the present study we use an MMP array to investigate the blood levels of several MMPs in stroke patients and its relation with brain tissue damage and neurological outcome. Methods— Twenty-four patients with middle cerebral artery occlusion who received thrombolytic therapy were included. Blood samples were drawn before tissue plasminogen activator treatment and an MMP array (multiplex enzyme-linked immunosorbent assay [ELISA]) was performed including gelatinases (MMP-2 and MMP-9), collagenases (MMP-1, MMP-8, and MMP-13), stromelysines (MMP-3 and MMP-10), and MMP endogen inhibitors (TIMP-1 and TIMP-2). To assess tissue lesion a serial multimodal MRI study was performed (pretreatment and at 24 hours). Results— Neither initial diffusion lesion nor hypoperfused volume was associated with metalloproteinase expression within the first 3 hours after stroke onset. Nevertheless, a strong correlation was found between MMP-9 and MMP-13 with diffusion-weighted image (DWI) lesion expansion (r=0.54, P=0.05 and r=0.60, P=0.017, respectively). Baseline levels of both MMP-9 (OR, 14;95% CI, 1.5 to 131; P=0.019) and MMP-13 (OR, 73; 95% CI, 3.9 to 1388; P=0.004) were independent predictors of final increase in brain infarct volume at 24 hours. Conclusions— Our results demonstrate that within the neuroinflammatory response, high levels of MMP-9 and MMP-13 are involved in DWI lesion growth despite thrombolytic therapy, suggesting its ultra-early role in brain injury.

Tissue plasminogen activator (t‐PA) promotes neutrophil degranulation and MMP‐9 release

Recombinant tissue plasminogen activator (t‐PA), the only approved stroke treatment, is used for clot lysis within the occluded brain artery. Unfortunately, matrix metalloproteinase‐9 (MMP‐9) concentration increases after t‐PA treatment and has been related to hemorrhagic transformation after ischemic stroke. Although the exact cellular source of brain MMP‐9 remains unknown, invading, inflammatory cells, such as neutrophils, release MMP‐9 to cross the blood brain barrier. Therefore, we hypothesize that the most feared side effect of stroke reperfusion therapy, brain hemorrhage, is related to t‐PA‐induced MMP‐9 release by neutrophils. We show by means of ELISA that t‐PA treatment promotes MMP‐9, MMP‐8, and tissue inhibitor metalloproteinase‐2 release from human neutrophils ex vivo within 10 and 30 min. Moreover, by zymography and Western blot, we observed that neutrophils are emptied of MMP‐9 content after t‐PA treatment at those times. Finally, total internal reflection fluorescent imaging allowed us to observe the t‐PA effect on neutrophils, showing the promotion of degranulation on these cells in vivo. Our data suggest that neutrophils are good candidates to be the main source of MMP‐9 following t‐PA stroke treatment and in consequence, partially responsible for thrombolysis‐related brain bleedings.

Poststroke C-Reactive Protein Is a Powerful Prognostic Tool Among Candidates for Thrombolysis

Background and Purpose— After acute stroke, an increased level of C-reactive protein (CRP) measured at discharge predicts unfavorable outcome. We sought to investigate whether CRP measured before tissue plasminogen activator (tPA) treatments may add prognostic information to guide stroke thrombolysis. Methods— Our target was 151 consecutive patients with an ischemic stroke involving the middle cerebral artery territory who received tPA within 3 hours of symptom onset. High-sensitivity CRP was measured before tPA administration, and CRP gene polymorphisms were determined (G1059C and C1444T). Functional outcome was evaluated by 3-month modified Rankin Scale (mRS). Results— A total of 143 tPA-treated patients were valid for analyses after exclusion of those with inflammatory diseases and those probably infected (CRP >6 mg/dL). Patients with history of previous stroke, hypertension, or atrial fibrillation had higher levels of CRP (P<0.05). CRP was higher in patients who died after thrombolysis (n=19) than in survivors (0.85 versus 0.53 mg/dL; P=0.002). Among the 94 patients with proximal middle cerebral artery occlusions, CRP level was 0.53 for 81 survivors versus 0.81 mg/dL for 13 who died (P=0.001). CRP-survival association was found even among patients who recanalized by the end of tPA infusion (P=0.007). A correlation between CRP and mRS was found (r=0.36, P=0.02), although CRP polymorphisms were not related to neurological outcome. In a logistic regression model, CRP (odds ratio=8.51; 95% CI, 2.16 to 33.5; P=0.002) and age (odds ratio=6.25; 95% CI, 1.44 to 27.19; P=0.015) were the only baseline mortality predictors. Conclusions— Admission CRP predicts mortality among tPA-treated stroke patients. Very early recanalization does not ameliorate the negative prognostic impact of elevated CRP.

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.

Vascular MMP-9/TIMP-2 and neuronal MMP-10 up-regulation in human brain after stroke: a combined laser microdissection and protein array study.

Matrix Metalloproteinases (MMPs) play an important role in brain injury after ischemic stroke. In the present study, we aimed to assess the global expression of MMP-Family proteins in the human brain after stroke by using a combination of Searchlight Protein Array and Laser Microdissection to determine their cellular origin. This study demonstrated that MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, MMP-13, and TIMP-1 were upregulated in the infarcted tissue compared to healthy control areas. Using laser microdissection we obtained specific neuronal and vascular populations from both infarcted and control areas. From these fractions, we showed that MMP-9 and TIMP-2 were highly produced in brain microvessels while MMP-10 was notably increased in neurons of the ischemic brain but not in healthy areas. These findings demonstrate a selective cell-dependent MMP secretion, opening the possibility of selectively targeting specific MMPs for neuroprotection or vasculoprotection following stroke.

Progression of Symptomatic Intracranial Large Artery Atherosclerosis Is Associated With a Proinflammatory State and Impaired Fibrinolysis

Background and Purpose— The molecular pathways involved in the progression of intracranial large artery atherosclerosis (ILA) are largely unknown. Our objective was to prospectively study the relationship between circulating levels of inflammatory markers and fibrinolysis inhibitors, and the risk of progression of symptomatic ILA. Methods— Seventy-five consecutive patients with first-ever symptomatic intracranial atherostenosis were studied. Blood levels of C-reactive protein (CRP), E-selectin, monocyte chemoattractant protein-1, intercellular adhesion molecule-1, matrix metalloproteinases 1, 2, 3, 8, 9, 10, and 13, plasminogen activator inhibitor-1 (PAI-1), and lipoprotein(a) were measured 3 months after the qualifying stroke or transient ischemic attack. Thereafter, patients underwent long-term transcranial Doppler follow-up to detect progression of ILA. Results— During a median follow-up time of 23 months, 25 (33%) patients showed ILA progression. Multivariable adjusted Cox regression models and Kaplan–Meier curves showed that high baseline level of CRP, E-selectin, intercellular adhesion molecule-1, matrix metalloproteinase 9, PAI-1, and lipoprotein(a) predicted ILA progression independently of vascular risk factors. Of them, only CRP (CRP>5.5 mg/L; HR, 5.4 [2.3 to 12.7]; P=0.0001) and PAI-1 (PAI-1>23.1 ng/mL; HR, 2.4 [1.0 to 5.8]; P=0.05) predicted ILA progression also independently of the other studied molecules. Conclusion— Progression of symptomatic ILA is associated with a proinflammatory state, as reflected by high levels of inflammatory markers, and with defective fibrinolysis, as indicated by raised concentrations of endogenous fibrinolysis inhibitors.

Factors Secreted by Endothelial Progenitor Cells Enhance Neurorepair Responses after Cerebral Ischemia in Mice

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.

Mobilization, endothelial differentiation and functional capacity of endothelial progenitor cells after ischemic stroke.

Endothelial progenitor cells (EPCs) have introduced new possibilities for cell-based vasculogenesis treatment after stroke. In this study we quantified circulating levels of EPCs in stroke patients and in healthy controls, and evaluated the potential of EPCs to induce vasculogenesis in vitro. Blood was drawn from tPA-treated stroke patients and control subjects, and the circulating EPCs levels in each group were quantified by flow cytometry and cell culture assays. Immunophenotyping was performed using multiple markers (UEA-lectin, CD133, vWF and KDR) and tubulogenic function was assessed with the Matrigel® assay. The produced angiogenic factors were quantified by multiple ELISA and RT-PCR. Fluorescence-activated cell sorting (FACS) revealed higher levels of circulating CD133+/CD34+/KDR+/CD45+ cells in the acute strokes as compared to the control subjects (p=0.02). On the other hand, more EPCs grew in cell culture from subacute strokes (p=0.016) than from controls. The endothelial and progenitor lineages of the EPCs were confirmed by immunophenotyping. Interestingly, the appearance of outgrowth EPCs (OECs) correlated positively to stroke severity (p=0.013). Finally, greater capacity to induce vasculogenesis in vitro was found in EPCs from subacute strokes (p=0.03), which we attribute to a higher expression and secretion of angiogenic factors. Our results suggest an early EPC mobilization but an enhanced angiogenic function in the subacute phase of stroke. Nonetheless, development of cell-based therapy for stroke will require further studies to identify those EPCs with the greatest therapeutic potential.

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.

Differentiating ischemic from hemorrhagic stroke using plasma biomarkers: The S100B/RAGE pathway

Although neuroimaging is useful in differentiating ischemic (IS) from hemorrhagic (ICH) stroke in the Emergency Department, a wide-available rapid biochemical test would add advantages in the pre-hospital triage and management of stroke patients. Our aim was to examine the predictive value of a panel of blood-borne biomarkers to differentiate IS from ICH. Admission blood samples obtained within 24h from stroke symptoms onset were tested by ELISA for CRP, D-dimer, sRAGE, MMP9, S100B, BNP, NT-3, caspase-3, chimerin-II, secretagogin, cerebellin and NPY. The complete protocol was achieved in 915 patients (776 IS, 139 ICH). Among blood samples obtained <6 h from symptoms onset (n=337), S100B levels were increased in ICH (107.58 vs 58.70 pg/mL; p<0.001) whereas sRAGE levels were decreased (0.77 vs 1.02 ng/mL; p=0.009) as compared to IS. In this subset of patients S100B (OR 3.97 95% CI 1.82-8.68; p=0.001) and sRAGE (OR 0.22 95% CI 0.10-0.52; p<0.001) were independently associated with ICH. A regression tree was created by CART method showing good classification ability (AUC=0.762). Similar results were found for samples obtained within 3 h. In conclusion, a combination of biomarkers including those of the S100B/RAGE pathway seems promising to achieve a rapid biochemical diagnosis of IS versus ICH in the first hours from symptoms onset. This article is part of a Special Issue entitled: Translational Proteomics.