Arantxa Ortega-Aznar

Increased Brain Expression of Matrix Metalloproteinase-9 After Ischemic and Hemorrhagic Human Stroke

Background and Purpose— Abnormal expression of some matrix metalloproteinases (MMP) has shown to play a deleterious role in brain injury in experimental models of cerebral ischemia. We aimed to investigate MMP-2 (gelatinase A) and MMP-9 (gelatinase B) in brain parenchyma in both ischemic and hemorrhagic strokes. Methods— Postmortem fresh brain tissue from 6 ischemic and 8 hemorrhagic stroke patients was obtained within the first 6 hours after death. Finally, 78 brain tissue samples from different areas (infarct, peri-infarct, perihematoma and contralateral hemisphere) were studied. To quantify gelatinase content we performed gelatin zymograms that were confirmed by Western Blot Analysis, immunohistochemistry to localize MMP source, and in situ zymography to detect gelatinase activity. Results— Among ischemic cases, gelatin zymography showed increased MMP-9 content in infarct core although peri-infarct tissue presented also higher levels than contralateral hemisphere (P<0.0001 and P=0.042, respectively). Within infarct core, MMP-9 was mainly located around blood vessels, associated to neutrophil infiltration and activated microglial cells. In peri-infarct areas the major source of MMP-9 were microglial cells. Tissue around intracranial hemorrhage also displayed higher MMP-9 levels than contralateral hemisphere (P=0.008) in close relationship with glial cells. MMP-2 was constitutively expressed and remained invariable in different brain areas. Conclusions— Our results demonstrate in situ higher levels of MMP-9 in human brain tissue after ischemic and hemorrhagic stroke, suggesting a contribution of MMP-9 to ischemic brain injury and perihematoma edema.

MMP-9–Positive Neutrophil Infiltration Is Associated to Blood–Brain Barrier Breakdown and Basal Lamina Type IV Collagen Degradation During Hemorrhagic Transformation After Human Ischemic Stroke

Background and Purpose— An abnormal expression of some matrix metalloproteinases (MMPs) is related with hemorrhagic transformation events after stroke. Our aim was to investigate MMP-2 and MMP-9 in the ischemic brain and its relation with blood–brain barrier breakdown after hemorrhagic transformation in human stroke. Methods— We assessed 5 cases of fatal ischemic strokes with hemorrhagic complications; brain samples were obtained from infarct, hemorrhagic, and contralateral tissue. MMP-9 and MMP-2 content was analyzed by zymography and immunohistochemistry was performed to localize MMP-9 and to assess collagen IV integrity in the basal lamina. Laser capture microdissection was performed to isolate blood–brain barrier vessels to study these MMPs. Results— Overall, MMP-9 levels were higher both in hemorrhagic and nonhemorrhagic infarcted tissue compared to contralateral areas (P<0.0001 and P<0.05). Moreover, levels of the cleaved MMP-9 85kDa-form were significantly elevated in the hemorrhagic compared to nonhemorrhagic and contralateral areas (P=0.033 and P<0.0001). No changes were found for MMP-2 content. Immunostaining revealed a strong MMP-9–positive neutrophil infiltration surrounding brain microvessels associated with severe basal lamina type IV collagen degradation and blood extravasation. Microdissection confirmed that content of MMP-9 was similarly high in microvessel endothelium from hemorrhagic and infarcted areas compared to contralateral hemisphere vessels (P<0.05), pointing to neutrophils surrounding dissected microvessels as the main source of MMP-9 in hemorrhagic areas. Conclusions— Our results show a strong neutrophil infiltration in the infarcted and hemorrhagic areas with local high MMP-9 content closely related to basal lamina collagen IV degradation and blood–brain barrier breakdown. Microvessel and inflammatory MMP-9 response are associated with hemorrhagic complications after stroke.

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.

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.

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.

Stroke and multi-infarct dementia as presenting symptoms of giant cell arteritis: report of 7 cases and review of the literature.

Cerebrovascular accidents (CVAs) and multi-infarct dementia have rarely been reported as presenting symptoms of giant cell arteritis (GCA), although 3%-4% of patients with GCA may present with CVAs during the course of the disease. We describe 7 patients with biopsy-proven GCA who presented with stroke or multi-infarct dementia. Most of them had other symptoms of GCA when the disease began that were misdiagnosed or not noticed. The internal carotid arteries were involved in 4 patients and the vertebrobasilar arteries in 3, with bilateral vertebral artery occlusion in 1. Small cerebral infarction foci on cranial computed tomography (CT) scan and magnetic resonance imaging (MRI) were found in 5 cases, and cerebellar infarction, in 2. MR angiography showed intracranial arteritis in 4 cases. Treatment with glucocorticoids and adjunctive antiplatelet or anticoagulant therapy was given in all cases, with neurologic improvement in 5. Two patients died. Necropsy demonstrated generalized GCA involving the medium and small cerebral vessels in 1 case. Central nervous system involvement is a rare complication in GCA but is important to recognize, as it can be reversible if diagnosed and treated promptly. Suspicion should arise in elderly patients suffering from strokes with a quickly progressing stepwise course and associated headache, fever, or inflammatory syndrome. In these cases, temporal artery biopsy should be performed without delay. Early diagnosis of GCA and immediate initiation of corticosteroid treatment may prevent progressive deterioration and death. Additional antiplatelet or anticoagulant therapy should be evaluated according to the individual risk and benefit to the patient under care.

Fas system activation in perihematomal areas after spontaneous intracerebral hemorrhage.

Background and Purpose— Apoptosis has been implicated as the prominent form of cell death in the brain perihematomal region in animal models and in autopsy or postsurgical human studies. Both the Fas system and caspase activation play a central role in apoptotic pathways. The aims of this study were to investigate soluble Fas (s-Fas) plasma levels after acute intracerebral hemorrhage (ICH), to determine its influence on clinical and radiologic features, and to assess Fas receptor and Fas ligand (Fas-L) protein expression in human ICH brain tissue. Methods— s-Fas plasma levels were determined on admission in 78 consecutive ICH patients and serially in a subgroup of 21 of them, at the time of neurologic assessment, by means of ELISA. ICH and perihematomal edema volumes were determined at baseline and on follow-up computed tomography scans, and ICH and perihematomal edema growth was calculated. The presence of Fas receptor and Fas-L was assessed in different brain tissue samples by immunoblotting from 6 deceased ICH patients and from 2 control subjects. Results— Mortality reached 20.5% of patients at the third month, and 48% of survivors had an unfavorable outcome (modified Rankin Scale score ≥3). The baseline s-Fas level in ICH patients was significantly lower than in healthy controls [160 (160–245) vs 269 (230–332) pg/mL, P<0.001], returning to normal values by 24 hours (P<0.05 for all determinations). Regarding radiologic features, the baseline s-Fas value was found to be inversely correlated to perihematomal edema growth at follow-up (r=−0.33, P=0.041). Finally, Fas-L content was highest in the perihematomal area compared with contralateral and remote ipsilateral areas in ICH patient and control samples. Conclusions— A decreased plasma s-Fas level together with an increased Fas-L amount in perihematomal brain tissue suggest Fas-mediated apoptosis involvement in this disease.

Brain Perihematoma Genomic Profile Following Spontaneous Human Intracerebral Hemorrhage

Background Spontaneous intracerebral hemorrhage (ICH) represents about 15% of all strokes and is associated with high mortality rates. Our aim was to identify the gene expression changes and biological pathways altered in the brain following ICH. Methodology/Principal Findings Twelve brain samples were obtained from four deceased patients who suffered an ICH including perihematomal tissue (PH) and the corresponding contralateral white (CW) and grey (CG) matter. Affymetrix GeneChip platform for analysis of over 47,000 transcripts was conducted. Microarray Analysis Suite 5.0 was used to process array images and the Ingenuity Pathway Analysis System was used to analyze biological mechanisms and functions of the genes. We identified 468 genes in the PH areas displaying a different expression pattern with a fold change between −3.74 and +5.16 when compared to the contralateral areas (291 overexpressed and 177 underexpressed). The top genes which appeared most significantly overexpressed in the PH areas codify for cytokines, chemokines, coagulation factors, cell growth and proliferation factors while the underexpressed codify for proteins involved in cell cycle or neurotrophins. Validation and replication studies at gene and protein level in brain samples confirmed microarray results. Conclusions The genomic responses identified in this study provide valuable information about potential biomarkers and target molecules altered in the perihematomal regions.

The proteome of human brain after ischemic stroke.

Although stroke is among the most common causes of death and chronic disability worldwide, the proteome of the ischemic human brain remains unknown. Only a few studies have investigated the ischemic brain proteome in rodent stroke models. We performed aproteomic study of the human brain after ischemic stroke usinga 2-dimensional differential gel electrophoresis-based proteomic approach. In brain samples from 6 deceased stroke patients and 3 control subjects, there was an average of 1,442 ± 231 protein spots in the gels. Changes of at least 1.5-fold in the relative expression of 132 protein spots between different cerebral areas (infarct core, peri-infarct, and contralateral tissue) were identified (p < 0.05); 39 of these were successfully identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Among the identified protein spots, we validated the results of 10 proteins by Western blot and determined the cellular localization in brain parenchyma for 3 of the identified proteins: dihydropyrimidinase-related protein 2, vesicle-fusing ATPase, and Rho dissociation inhibitor 1. These results contribute to understanding the processes that follow cerebral ischemia; moreover, some of the identified proteins may be therapeutic targets or biologic markers for determining the diagnosis and prognosis of stroke.