Simone Wagner

Profiles of Matrix Metalloproteinases, Their Inhibitors, and Laminin in Stroke Patients Influence of Different Therapies

BACKGROUND AND PURPOSE The goal of this study was to determine the temporal profile of several matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs), and laminin (an MMP substrate) in human stroke under different treatment paradigms, including thrombolysis and hypothermia. METHODS We serially measured the serum levels of MMP-2, MMP-3, MMP-9, MMP-13, TIMP-1, TIMP-2, and laminin in 50 patients with acute ischemic stroke using zymography or enzyme-linked immunosorbent assay. Patients were treated with heparin, therapeutic thrombolysis, or hypothermia. Scandinavian Stroke Scale scores were obtained at baseline. Infarct volume was measured with CT scanning on day 4 after stroke onset. Healthy persons were used as control subjects. RESULTS MMP-2 and MMP-9 increased during the course of ischemia, whereas intact laminin and TIMP-2 decreased significantly (P<0.05). MMP-9 and laminin levels varied significantly by infarct size (P=0.001) and therapy (P=0.0005). MMP-9 levels were significantly higher in patients treated with tissue plasminogen activator (tPA) compared with patients treated with hypothermia. The cleaved form of MMP-9 was found solely in 4 patients treated with tPA. Intact laminin levels were significantly lower in the tPA group than in the hypothermia group. CONCLUSIONS Selected MMPs and TIMPs are involved in the pathophysiology of acute stroke. This is also reflected by changes in laminin. Treatment paradigms differentially influence levels of MMP-9 and laminin. Combination therapies explicitly involving MMP inhibition could be of value in future treatment strategies.

Rapid Disruption of an Astrocyte Interaction With the Extracellular Matrix Mediated by Integrin α6β4 During Focal Cerebral Ischemia/Reperfusion

Background and Purpose Integrins participate in cerebral microvascular integrity and signaling during focal ischemia/reperfusion. The integrin subunits α1, α6, and β1 are distributed identically on normal cerebral microvessels. Studies in epithelium indicate that integrin α6β4, which interacts with laminin-5 in the basal lamina/extracellular matrix, is unique. This study describes the exact location of α6, β4, and α6β4 and that their responses in focal cerebral ischemia are relevant to astrocyte-matrix interactions. Methods The effect of middle cerebral artery occlusion and subsequent reperfusion on the microvascular expression of α6β4 and laminin-5 in regions of cellular injury (dUTP incorporation) was examined in 15 nonhuman primates. Well-characterized antibodies against human α6, β4, α6β4, laminin-5 and laminin-1, endothelial CD31, and vascular markers were measured with computerized video imaging and laser confocal microscopy. Results Integrin α6β4 was localized on astrocytes where it connects with the extracellular matrix at the astrocyte-vessel interface. It represented 59.3±16.4% of α6 antigen in cerebral microvessels <100 μm in diameter. By 2 hours of ischemia, the significant reduction in α6 expression (2 P <.001) was accompanied by decreases in β4/laminin-5 (0.76±0.03 to 0.20±0.09; 2 P =.001) and α6β4/laminin-5 (0.73±0.18 to 0.25±0.11; 2 P =.001) in the region of dUTP incorporation. Parallel changes in laminin-5 and laminin-1 were less pronounced and coincided by 24 hours. Conclusions This is the first description of a potential role of integrin α6β4 in the brain, where it mediates astrocyte-matrix interactions. The dramatic disappearance of α6β4 relative to its ligands reflects early loss of integrity between the astrocyte and the vessel wall in selected microvessels in response to ischemia.

Rapid Loss of Microvascular Integrin Expression During Focal Brain Ischemia Reflects Neuron Injury

The integrity of cerebral microvessels requires the close apposition of the endothelium to the astrocyte endfeet. Integrins α1β1 and α6β4 are cellular matrix receptors that may contribute to cerebral microvascular integrity. It has been hypothesized that focal ischemia alters integrin expression in a characteristic time-dependent manner consistent with neuron injury. The effects of middle cerebral artery occlusion (MCAO) and various periods of reperfusion on microvasclar integrin α1β1 and α6β4 expression were examined in the basal ganglia of 17 primates. Integrin subunits α1 and β1 colocalized with the endothelial cell antigen CD31 in nonischemic microvessels and with glial fibrillary acidic protein on astrocyte fibers. Rapid, simultaneous, and significant disappearance of both integrin α1 and β1 subunits and integrin α6β4 occurred by 2 hours MCAO, which was greatest in the region of neuron injury (ischemic core, Ic), and progressively less in the peripheral (Ip) and nonischemic regions (N). Transcription of subunit β1 mRNA on microvessels increased significantly in the Ic/Ip border and in multiple circular subregions within Ic. Microvascular integrin α1β1 and integrin α6β4 expression are rapidly and coordinately lost in Ic after MCAO. With loss of integrin α1β1, multiple regions of microvascular β1 mRNA up-regulation within Ic suggest that microvessel responses to focal ischemia are dynamic, and that multiple cores, not a single core, are generated. These changes imply that microvascular integrity is modified in a heterogeneous, but ordered pattern.

Minocycline and hypothermia for reperfusion injury after focal cerebral ischemia in the rat—Effects on BBB breakdown and MMP expression in the acute and subacute phase

Reperfusion injury is a complication of recanalization therapies after focal cerebral ischemia. The disruption of the blood-brain barrier (BBB) caused by up-regulated metalloproteinases (MMPs) can lead to edema and hemorrhage. Middle cerebral artery occlusion (MCAO=90 min) and reperfusion (R=24 h vs. 5 days) was induced in male Wistar rats. Rats were randomized in four groups: (1) control (C), (2) twice daily minocycline (30 mg/kg bodyweight) every day (M), (3) hypothermia (33 degrees C) for 4 h starting 60 min after occlusion (H), (4) combination of groups 2 and 3 (MH). Serial MRI was performed regarding infarct evolution and BBB disruption, MMP-2 and MMP-9 were assessed by zymography of serum and ischemic brain tissue, and a functional neuroscore was done at 24 h and 5 days. M and H reduced both infarct sizes, volume and signal intensity of BBB breakdown and improved neuroscore at all points in time to the same extent. This was most likely due to inhibition of MMP-2 and MMP-9. The presence of MMP-9 at 24 h or MMP-2 at 5 days in brain tissue correlated with BBB breakdown whereas serum MMP-2- and -9 showed no relationship with BBB breakdown. The combination MH had a small but not significantly additional effect over the single treatments. Minocycline seems to be as neuroprotective as hypothermia in the acute and subacute phase after cerebral ischemia. One essential mechanism is the inhibition of MMPs. The combination therapy is only slightly superior. The net effect of MMPs inhibition up to 5 days after focal cerebral ischemia is still beneficial.

Trends and Future Developments in the Pharmacological Treatment of Acute Ischaemic Stroke

SummaryStroke stands as the third leading cause of death. It makes great demands on patients, who must not only survive the complications of the acute stages, but must cope then with the great physical and economic costs of long-term disabilities. Therefore, there is urgent need to establish generally useful regimens for the acute treatment of ischaemic stroke. Three treatment approaches are based upon pathophysiologic concepts derived from experimental work with focal cerebral ischaemia. These include pharmacologic strategies for arterial recanalisation, inhibition of inflammatory processes and neural protection.Focal cerebral ischaemia secondary to occlusion of a brain-supplying artery initiates neuronal and microvascular events, and the simultaneous processes of inflammation which further injure tissue. The use of plasminogen activators to mediate thrombus and lysis in the acute setting has been shown to be clinically beneficial. Further work with arterial reperfusion strategies is under way. Early clinical studies with polymorphonuclear leukocyte-dependent endothelial adhesion receptor antagonists are being completed, but a strategy has yet to emerge. A large effort examining the potential efficacy of agents which may stabilise or protect neurons from ischaemic injury has shown promise in experimental models, and has been translated into clinical trials.Experimental work, and limited clinical experience, have indicated that: (a) the time window for intervention is important in limiting ischaemic and inflammatory injury, and for reducing the risk of haemorrhagic transformation; (b) putative neuroprotective strategies may potentially elongate the time interval for treatment; and (c) limitations from the adverse effects of plasminogen activators and of agents which beneficially affect neuronal dysfunction during ischaemia must yet be overcome. This review surveys pharmacological approaches currently undergoing evaluation which provide the goal of establishing effective strategies for the treatment of patients with acute cerebral ischaemia.

Topographically graded postischemic presence of metalloproteinases is inhibited by hypothermia.

To test the hypothesis that presence of metalloproteases (MMPs), their inhibitors (TIMPs) and their substrate laminin-5 differs between the ischemic core and the surrounding tissue, we examined the impact of middle cerebral artery occlusion/reperfusion (MCA:O/R) on these molecules in different regions of the infarct. We also investigated the influence of hypothermia on the progression of the ischemic lesion and MMP activity. Brain sections from 64 Wistar rats subjected to MCA:O/R were examined by means of cytohistochemistry and zymography. The artery was occluded for 2 h followed by 3, 5, 8 and 12 h of reperfusion. Well characterized antibodies against laminin-5, MMPs and TIMP-2 were used. A total of 32 rats were treated with hypothermia. The presence of each antigen was related to the following regions of interest: ischemic core with BBB breakdown (I(c)), surrounding ischemic tissue without BBB breakdown (I(r)), and the contralateral non-ischemic region (N). Regions of interest were defined by MRI. The I(c) increased over time at the cost of the I(r). BBB breakdown occurred early in the ischemic core and increased over time. Hypothermia reduced the BBB breakdown at all time points. A graded decreased presence of laminin-5 was observed with 16.5+/-3.7(N)>10+/-2.8(I(r))>4+/-1.4(I(c)) immunopositive microvessels/mm(2) at 3 h of reperfusion. MMP-9 showed a reverse pattern with 0 (N)<4+/-0.8(I(r))<10+/-1.5(I(c)) immunopositive microvessels/mm(2). Hypothermia decreased the MMP activity measured by zymography. Laminin-5 and MMP presence relate directly to the degree of postischemic injury. Hypothermia reduces the conversion from the I(r) to ischemic core and the degree of BBB as well as MMP abundance.

Spontaneous Resolution of a Large Spinal Epidural Hematoma: Case Report

Spontaneous spinal epidural hematoma is a rare condition that usually requires surgical evacuation of the hematoma. We report a case of spontaneous spinal epidural hematoma that was probably associated with aspirin intake. The initial clinical signs and symptoms included sharp, left-sided neck pain and weakness of the left arm. The initial magnetic resonance image showed a spinal epidural hematoma extending from C2 to C6, with compression of the myelon. This case is remarkable for dramatic clinical improvement within 12 hours and the magnetic resonance imaging documentation of complete resolution within 3 days. For each patient with a stable or improving neurological status, conservative management monitored by magnetic resonance imaging might be the treatment of choice.

Matrix metalloproteinases in peripheral blood and cerebrospinal fluid in patients with Alzheimer's disease

BACKGROUND Deposition of amyloid beta in senile plaques and in cerebral blood vessels is one hallmark of the pathogenesis of Alzheimers disease (AD). The ability of several matrix metalloproteinases (MMPs) to degrade amyloid precursor protein leading to aggregation of amyloid beta, as well as the increased expression of MMPs in post mortem brain tissue of Alzheimers patients, indicate that MMPs play an important role in the pathogenesis of AD. METHODS We investigated levels of MMP-2,-3,-9 and -10 in plasma and cerebrospinal fluid (CSF) of AD patients (n = 14) by gelatin and casein zymography. Comparisons between AD patients and controls relative to levels of MMP-2, MMP-3, MMP-9, and MMP-10 were made with Wilcoxon rank statistics. Pearson correlations were computed as measures of association. RESULTS MMP-3 in AD was significantly elevated in plasma (p = 0.006) and there was a trend towards increase in CSF (p = 0.05). MMP-2 in CSF of AD patients was significantly decreased (p = 0.02) while levels in plasma remained unchanged. MMP-9 and MMP-10 could not be detected in CSF; MMP-10 was unchanged in plasma, but MMP-9 was significantly decreased (p = 0.02). CONCLUSIONS These findings constitute further evidence for the important role of MMPs in the pathogenesis of AD.

Gene expression in human peripheral blood mononuclear cells upon acute ischemic stroke

AbstractBackground and purposeIschemic stroke provokes a systemic inflammatory response. The purpose of this study was to characterize this response on the gene expression level in circulating mononuclear leukocytes from acute ischemic stroke (AIS) patients.MethodsRNA from peripheral blood mononuclear cells (PBMCs) of AIS patients (24 + 2 hours after onset of symptoms) was analyzed with Affymetrix U133A GeneChips using a pooled design. We compared the gene expression signature from AIS patients (n = 20), stroke survivors (n = 15), patients with acute traumatic brain injury (ATBI, n = 15) and healthy control subjects without vascular risk factors (n = 15).ResultsExpression levels of 9682 probe sets with present calls on each GeneChip were compared. Between AIS patients and stroke survivors or between AIS patients and ATBI patients there were no significant differences in expression values of single genes after correction for multiple testing. However, comparison of the PBMC expression profiles from AIS patients and healthy subjects revealed significantly different expression (p = 0.012) of a single probe set, specific for phosphodiesterase 4 D (PDE4D). In order to detect modest expression differences in multiple genes with a presumed cumulative effect we studied the gene expression of functional groups of genes by global statistical tests. Analysis of 11 gene groups revealed differential expression between AIS patients and healthy subjects for genes involved in the inflammatory response (GeneOntology GO:0006954). Genes encoding the N-formyl peptide receptor-like 1 (FPRL1), interleukin-1 receptor antagonist (IL1RN) and complement component 3a receptor 1 (C3AR1) contributed most to the observed difference.ConclusionsThis transcriptome analysis did not identify significant changes between circulating mononuclear cells from AIS patients 24 hours after stroke and closely matched stroke survivors. However, comparing AIS patients with healthy control subjects revealed measurable differences in PDE4D and in inflammatory response genes when considered as a set.

MMP-2 and MMP-9 levels in peripheral blood after subarachnoid hemorrhage.

MMPs play an important role in ischemic and hemorrhagic stroke. We analyzed replicate serum samples from 20 normal healthy individuals to assess reproducibility of MMP determinations, and found that MMP-2 and MMP-9 determinations were quite consistent. We then studied the serum levels of MMP-2 and MMP-9 in patients suffering from subarachnoid hemorrhage (SAH), another stroke subtype. Serum MMP-2 and MMP-9 levels from SAH patients were measured sequentially using gelatine zymography in 11 patients after acute SAH. The occurrence of intracerebral aneurysms and vasospasms and the initial Hunt and Hess score were analysed in relation to MMP-levels. MMP-2 levels are significantly decreased while MMP-9 levels are increased in SAH patients relative to controls. MMP-2 levels remain depressed out to day 12 post SAH, but MMP-9 levels may recover by day 12.