Lidia García-Bonilla

Metalloproteinase and stroke infarct size: role for anti-inflammatory treatment?

Deregulation of matrix metalloproteinases (MMPs), the largest class of human proteases, has been implicated in brain damage in both animal and human studies. Some MMPs are elevated after stroke (both in plasma and in brain tissue), and their expression is enhanced by t‐PA during thrombolysis related to hemorrhagic transformation events. Although the exact cellular source of MMPs remains unknown, brain endothelium, astrocytes, neurons, and inflammatory‐activated cells, such as neutrophils, may release MMP‐2, MMP‐3, MMP‐8, MMP‐9, MMP‐10, and/or MMP‐13. Neurovascular perturbations occurring after stroke lead to blood–brain barrier leakage, edema, hemorrhage, leukocyte infiltration, and progressive inflammatory reactions to brain injury over hours or even days after the initial stroke. Synthesized MMP inhibitors and several compounds used for stroke secondary prevention, such as anti‐inflammatory drugs, might decrease MMPs and improve the acute treatment of human brain ischemia without compromising the beneficial effects of matrix plasticity during stroke recovery.

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.

Matrix metalloproteinase-13 is activated and is found in the nucleus of neural cells after cerebral ischemia.

Matrix metalloproteinases (MMPs) have been implicated in the pathophysiology of ischemic stroke. In this study, we investigated the time course of gelatinolytic activation in a rat model of permanent ischemia. We observed an activation of MMPs as early as 30 mins after the ischemic insult, mainly in the nuclei of brain cells. Besides, we explored MMP-13 expression in brain samples of the animal model and stroke deceased patients. We observed an upregulation of active MMP-13 in rat brains (P< 0.05) after 90 mins of cerebral ischemia. Human infarct/periinfarct samples also showed higher levels of active MMP-13 (P< 0.05) compared with contralateral ones. Interestingly, we found that MMP-13 colocalized with 46-diamidino-2-phenyl indole signal by immunohistochemistry in both humans and rats, suggesting an intranuclear localization for MMP-13. Immunohistochemistry also revealed that MMP-13 was mainly produced by neurons, in both species, but also by oligodendrocytes in rats, and by astrocytes in humans. Finally we subjected a rat primary neuronal culture to oxygen and glucose deprivation (OGD) and we reproduced the nuclear translocation of MMP-13 in vitro. Nuclear extracts from cells confirmed upregulation of active MMP-13 after OGD (P< 0.05). These results suggest that MMP-13 activation and its nuclear translocation is an early consequence of an ischemic stimulus.

Plasma VAP-1/SSAO activity predicts intracranial hemorrhages and adverse neurological outcome after tissue plasminogen activator treatment in stroke.

Background and Purpose— Vascular adhesion protein-1 (VAP-1) is a cell surface and circulating enzyme involved in recruitment of lymphocytes and neutrophils through its semicarbazide-sensitive amine oxidase (SSAO) activity. We aimed to study plasma VAP-1/SSAO activity in relation to the risk for intracranial bleeding complications in patients with stroke treated with tissue plasminogen activator (tPA), the greatest safety concern with this treatment. Methods— In 141 patients with ischemic stroke, we measured VAP-1/SSAO activity in plasma taken before tPA administration. Hemorrhagic events were classified according to brain CT criteria and functional outcomes evaluated using the National Institutes of Health Stroke Scale. We also assessed the potential therapeutic effect of blocking VAP-1/SSAO activity in a rat embolic stroke model treated with tPA. Results— We saw significantly higher levels of plasma VAP-1/SSAO activity in patients who subsequently experienced hemorrhagic transformation. Elevated plasma VAP-1/SSAO activity also predicted worse neurological outcome in these patients. In the rat model, we confirmed that use of the inhibitor semicarbazide prevented adverse effects caused by delayed tPA administration, leading to a smaller infarct volume. Conclusions— Our data demonstrate that baseline VAP-1/SSAO activity predicts parenchymal hemorrhage after tPA, suggesting the safety of thrombolytic agents could be improved by considering VAP-1/SSAO activity. Furthermore, anti-VAP-1/SSAO drugs given with tPA may prevent neurological worsening in patients with ischemic stroke.

Evidence for the efficacy of statins in animal stroke models: a meta-analysis

J. Neurochem. (2012) 122, 233–243.

VAP-1/SSAO Plasma Activity and Brain Expression in Human Hemorrhagic Stroke

Background: Vascular adhesion protein-1 (VAP-1) is a cell surface and circulating enzyme that belongs to the semicarbazide-sensitive amine oxidase (SSAO) family, which oxidatively deaminates primary amines and is implicated in leukocyte extravasation. Our aim was to investigate the alteration of soluble VAP-1/SSAO activity in plasma samples after acute intracerebral hemorrhage (ICH) and its presence in human ICH brain tissue. Methods: VAP-1/SSAO activity was determined in plasma of 66 ICH patients and 58 healthy controls. In addition, we assessed the expression of VAP-1/SSAO in postmortem brain tissue from hemorrhagic stroke patients by Western blot and immunohistochemistry. Results: We observed significantly higher levels of plasma VAP-1/SSAO activity in patients with ICH compared to matched elderly controls (p = 0.001). Plasma VAP-1/SSAO activity <2.7 pmol/min·mg and baseline ICH volume <17 ml were independent predictors of neurological improvement after 48 h (OR 6.8, 95% CI 1.14–41.67, p = 0.035, and OR 10.64, 95% CI 1.1–100, p = 0.041, respectively), after adjustment for baseline stroke severity. We also found that membrane-bound VAP-1/SSAO levels were lower in the perihematoma region than in the corresponding contralateral brain areas of patients deceased due to ICH (p = 0.024). Conclusions: Our data demonstrate that plasma VAP-1/SSAO activity is increased in ICH and predicts neurological outcome, suggesting a possible contribution of the soluble protein in secondary brain damage. Furthermore, anti-VAP-1/SSAO strategies might be a promising approach to prevent neurological worsening following ICH.

Effects of acute post-treatment with dipyridamole in a rat model of focal cerebral ischemia

Dipyridamole (DP) is a platelet inhibitor with known antithrombotic benefits in stroke prevention. In addition to its anti-aggregant properties, recent studies have reported that DP promotes anti-inflammatory, anti-oxidative and neuroprotective effects. We aimed to test whether post-treatment with DP may exert protection after ischemic cerebral injury in the rat. For this purpose, rats were subjected to 120 min or 90 min of middle cerebral artery occlusion (MCAO) followed by 24 or 48 h of reperfusion, respectively. Either DP (100mg/kg) or vehicle was administered i.v. at the onset of reperfusion; rats subjected to 90 min MCAO also received additional doses of DP orally (60 mg/kg) at 24 and 36 h after ischemia. Matrix metalloproteinases, extravasated hemoglobin content and IL-6, MIP-1α and MCP-1 cytokine level were examined in brain tissue by zymography, western blot and multiple ELISA, respectively. DP post-treatment led to a neurological improvement in both models (p < 0.05) and a significant reduction in the infarct volume of rats subjected to 90 min of ischemia, as compared to vehicle group (7.9% vs. 24.4%, p = 0.03). This neuroprotection was accompanied by a modest increase in expression of MMP-9 pro-form and a significant attenuation of MIP-1α levels in the infarcted hemisphere. These results provide support for the development of novel therapies based on DP for acute treatment of stroke. In selected animals, intravenous administration of high dose DP induced an adverse hypotensive effect leading to rapid death. Thus, alternative ways of acute administration must be examined in order to avoid this unfavorable effect.

A new method for focal transient cerebral ischaemia by distal compression of the middle cerebral artery

A. Morancho, L. García‐Bonilla, V. Barceló, D. Giralt, M. Campos‐Martorell, S. Garcia, J. Montaner and A. Rosell (2012) Neuropathology and Applied Neurobiology38, 617–627

Regulatory proteins of eukaryotic initiation factor 2-alpha subunit (eIF2α) phosphatase, under ischemic reperfusion and tolerance

Phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α), which is one of the substrates of protein phosphatase 1 (PP1), occurs rapidly during the first minutes of post‐ischemic reperfusion after an episode of cerebral ischemia. In the present work, two experimental models of transient global ischemia and ischemic tolerance (IT) were used to study PP1 interacting/regulatory proteins following ischemic reperfusion. For that purpose we utilized PP1 purified by microcystin chromatography, as well as 2D DIGE of PP1α and PP1γ immunoprecipitates. The highest levels of phosphorylated eIF2α found after 30 min reperfusion in rats without IT, correlated with increased levels in PP1 immunoprecipitates of the inhibitor DARPP32 as well as GRP78 and HSC70 proteins. After 4 h reperfusion, the levels of these proteins in PP1c complexes had returned to control values, in parallel to a significant decrease in eIF2α phosphorylated levels. IT that promoted a decrease in eIF2α phosphorylated levels after 30 min reperfusion induced the association of GADD34 with PP1c, while prevented that of DARPP32, GRP78, and HSC70. Different levels of HSC70 and DARPP32 associated with PP1α and PP1γ isoforms, whereas GRP78 was only detected in PP1γ immunoprecipitates. Here we suggest that PP1, through different signaling complexes with their interacting proteins, may modulate the eIF2α phosphorylation/dephosphorylation during reperfusion after a transient global ischemia in the rat brain. Of particular interest is the potential role of GADD34/PP1c complexes after tolerance acquisition.

Neuronal TIMP-1 Release Accompanies Astrocytic MMP-9 Secretion and Enhances Astrocyte Proliferation Induced by β-Amyloid 25-35 Fragment

The neuropathology of Alzheimers disease (AD) is accompanied by an inflammatory response that includes neurodegeneration and glial reactivity. Tissue remodeling proteins, such as matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs), are inflammatory mediators that might play a dual role in the AD brain. We aimed to investigate the effects of β‐amyloid (Abeta) on the MMP‐9/TIMP‐1 balance and its involvement in Abeta toxicity in neurons and glial cells. Our results demonstrate that the neurotoxic 25–35 Abeta fragment induces the activation of MMP‐9 and the increase of proMMP‐2/9 secretion and promotes the release of TIMP‐1 in a mixed cortical neuroglial culture. The same treatments performed in pure neuronal or astrocytic cultures confirm that astroglial cells are the major source of MMP‐9, whereas increased TIMP‐1 levels have a neuronal origin. Moreover, 25–35 Abeta fragment not only induced a release of these molecules but also caused expressional changes in MMP‐9 and TIMP‐1, correlated with the neurotoxicity process. We also show that TIMP‐1 promoted cell proliferation in a mixed neuroglial culture, and we confirm this effect in primary cultured astrocytes induced by rTIMP‐1 and 25–35 Abeta. Because the proliferative effect caused by Abeta 25–35 was enhanced by the presence of TIMP‐1, we suggest that the astroglial reactivity induced by chronic exposure of the peptide might be mediated in part by TIMP‐1, which is secreted mainly by injured neurons. In conclusion, our data suggest that the Abeta 25–35 fragment stimulates the MMP‐9–TIMP‐1 pathway, promoting gliosis, in a self‐defensive attempt to eliminate amyloid deposition from AD brains.