Somatic Gain of KRAS Function in the Endothelium Is Sufficient to Cause Vascular Malformations That Require MEK but Not PI3K Signaling.

RATIONALE: We previously identified somatic activating mutations in the KRAS (Kirsten rat sarcoma viral oncogene homologue) gene in the endothelium of the majority of human sporadic brain arteriovenous malformations; a disorder characterized by direct connections between arteries and veins. However, whether this genetic abnormality alone is sufficient for lesion formation, as well as how active KRAS signaling contributes to arteriovenous malformations, remains unknown. OBJECTIVE: To establish the first in vivo models of somatic KRAS gain of function in the endothelium in both mice and zebrafish to directly observe the phenotypic consequences of constitutive KRAS activity at a cellular level in vivo, and to test potential therapeutic interventions for arteriovenous malformations. METHODS AND RESULTS: Using both postnatal and adult mice, as well as embryonic zebrafish, we demonstrate that endothelial-specific gain of function mutations in Kras (G12D or G12V) are sufficient to induce brain arteriovenous malformations. Active KRAS signaling leads to altered endothelial cell morphogenesis and increased cell size, ectopic sprouting, expanded vessel lumen diameter, and direct connections between arteries and veins. Furthermore, we show that these lesions are not associated with altered endothelial growth dynamics or a lack of proper arteriovenous identity but instead seem to feature exuberant angiogenic signaling. Finally, we demonstrate that KRAS-dependent arteriovenous malformations in zebrafish are refractory to inhibition of the downstream effector PI3K but instead require active MEK (mitogen-activated protein kinase kinase 1) signaling. CONCLUSIONS: We demonstrate that active KRAS expression in the endothelium is sufficient for brain arteriovenous malformations, even in the setting of uninjured adult vasculature. Furthermore, the finding that KRAS-dependent lesions are reversible in zebrafish suggests that MEK inhibition may represent a promising therapeutic treatment for arteriovenous malformation patients. Graphical Abstract: A graphical abstract is available for this article.

Inhibition of Src family kinases improves cognitive function after intraventricular hemorrhage or intraventricular thrombin.

Intraventricular hemorrhage causes spatial memory loss, but the mechanism remains unknown. Our recent studies demonstrated that traumatic brain injury activates Src family kinases, which cause spatial memory loss. To test whether the spatial memory loss was due to blood in the ventricles, which activated Src family kinases, we infused autologous whole blood or thrombin into the lateral ventricles of adult rats to model non-traumatic intraventricular hemorrhage. Hippocampal neuron loss was examined 1 day to 5 weeks later. Spatial memory function was assessed 29 to 33 days later using the Morris water maze. Five weeks after the ventricular injections of blood or thrombin, there was death of most hippocampal neurons and significant memory deficits compared with sham operated controls. These data show that intraventricular thrombin is sufficient to kill hippocampal neurons and produce spatial memory loss. In addition, systemic administration of the non-specific Src family kinase inhibitor PP2 or intraventricular injection of siRNA-Fyn, a Src family kinase family member, prevented hippocampal neuronal loss and spatial memory deficits following intraventricular hemorrhage. The data support the conclusions that thrombin mediates the hippocampal neuronal cell death and spatial memory deficits produced by intraventricular blood and that these can be blocked by non-specific inhibition of Src family kinases or by inhibiting Fyn.

Significance of cyclooxygenase-2 elevation in middle cerebral artery for patients with hemorrhagic moyamoya disease.

The etiology and pathogenesis of moyamoya disease (MMD) remain elusive. Some inflammatory proteins, such as cyclooxygenase (COX)-2, are believed to be implicated in the development of MMD. So far, the relationship between COX-2 and MMD is poorly understood and reports on the intracranial vessels of MMD patients are scanty. In this study, tiny pieces of middle cerebral artery (MCA) and superficial temporal artery (STA) from 13 MMD patients were surgically harvested. The MCA and STA samples from 5 control patients were also collected by using the same technique. The expression of COX-2 was immunohistochemically detected and the average absorbance (A) of positively-stained areas was measured. High-level COX-2 expression was found in all layers of the MCA samples from all 5 hemorrhagic MMD patients, while positive but weak expression of COX-2 was observed only in the endothelial layer of the MCA samples from most ischemic MMD patients (6/8, 75%). The average A values of COX-2 in the hemorrhagic MMD patients were substantially higher than those in their ischemic counterparts (t=4.632, P=0.001). There was no significant difference in the COX-2 expression among the "gender" groups, or "radiographic grade" groups, or "lesion location" groups (P>0.05 for all). The COX-2 expression was detected neither in the MCA samples from the controls nor in all STA specimens. Our results suggested that COX-2 was up-regulated in the MCA of MMD patients, especially in hemorrhagic MMD patients. We are led to speculate that COX-2 may be involved in the pathogenesis of MMD and even contribute to the hemorrhagic stroke of MMD patients.

Association of ACE gene I/D polymorphism and ACE levels with hemorrhagic stroke: comparison with ischemic stroke.

In the present study, we investigated the association of insertion/deletion polymorphism of ACE gene with genetic predisposition to hemorrhagic stroke and also determined the mean ACE activity levels in ischemic and hemorrhagic stroke patients. Two hundred hemorrhagic stroke, 200 ischemic stroke patients and 200 gender and age matched controls were recruited for the study. We found statistically significant difference in the genotypic distribution between hemorrhagic patients and controls for dominant, co-dominant and recessive models. Significant difference was observed in the allelic frequencies between hemorrhagic patients and controls. Multiple logistic regression analysis confirmed these findings [adjusted OR for DD genotype was 2.46 (95 % CI 1.43-4.21) and p = 0.001] and [adjusted OR for ID genotype was 5.45 (95 % CI 2.6-10.4) and p = 0.001]. We have already established the association of this polymorphism in ischemic stroke patients. Comparing hemorrhagic with ischemic stroke, we found a significant difference in genotypic distribution between the two [for II vs. DD, χ (2) = 4.75; p = 0.03, OR = 0.5 (95 % CI 0.27-0.93) and for DD vs. ID, χ (2) = 5.1; p = 0.02, OR = 1.8 (95 % CI 1.1-3.3)]. Our results indicate that DD genotype and D allele are important risk factors for the development of stroke. Individuals harboring DD genotype of ACE I/D polymorphism are more predisposed to hemorrhagic stroke than ischemic stroke. Further, the mean ACE activity level was found to be significantly higher in hemorrhagic and ischemic stroke in comparison with controls, but there was no significant difference in the levels found between the two types of stroke.

The effects of previous statin treatment on plasma matrix metalloproteinase-9 level in Chinese stroke patients undergoing thrombolysis.

It is unclear whether previous statin therapy influences the prognosis, hemorrhagic transformation, and plasma matrix metalloproteinases (MMP)-9 levels in Chinese stroke patients receiving intravenous thrombolysis. We conduct a prospective cohort study of 193 patients treated with intravenous thrombolysis. All the enrolled patients were divided into 2 groups (the control group and the statin group), according to the previous history of statin use. The plasma MMP-9 levels were detected before and at 6 hours, 12 hours, 24 hours, and 72 hours after intravenous thrombolysis. The clinical outcome of stroke was measured in terms of the functional outcome and occurrence of symptomatic intracerebral hemorrhage. The MMP-9 levels increased after thrombolysis in statin group and control group. No significant intergroup difference was found in the MMP-9 levels before and at 6 hours after thrombolysis, but the levels were significantly lower in the statin group than in the control group at 12, 24, and 72 hours (P < .001) after thrombolysis. Similarly, no significant intergroup difference was noted in the occurrence of symptomatic intracranial hemorrhage as was the case with the modified Rankin scale (assessed by the Mann-Whitney U test) at 7 days (P = .428) and 90 days (P = .419) after thrombolysis. Our results indicate that pretreatment with statin can inhibit the thrombolysis-induced increase in plasma MMP-9 levels but does not significantly affect the prognosis of acute ischemic stroke patients undergoing intravenous thrombolysis.

Lipoprotein-associated phospholipase A2 during the hyperacute stage of ischemic and hemorrhagic strokes.

BACKGROUND: The objectives of the study were to compare lipoprotein-associated phospholipase A2 (Lp-PLA2) levels in a prospective cohort including both ischemic and hemorrhagic strokes at the hyperacute phase, and to investigate if these levels were associated with stroke severity. MATERIALS AND METHODS: Lp-PLA2 mass and activity were measured during the first 6 hours of symptom onset before any therapeutic intervention. The Lp-PLA2 level was analyzed by comparing the mass and activities in ischemic strokes and spontaneous intracerebral hemorrhages (ICH). Correlations between Lp-PLA2 levels and clinical scores as well as stroke volumes were made. The temporal evolution of Lp-PLA2 during the first week was analyzed in ischemic stroke patients. RESULTS: Lp-PLA2 mass was higher in ICH than in ischemic stroke (P = .001). Lp-PLA2 activity at admission correlated with initial and follow-up stroke volume in ICH (P = .003 and P = .004, respectively) but not in ischemic stroke. None of the measurements correlated with clinical severity for either diagnosis. Lp-PLA2 mass decreased during the first week after the use of statins in ischemic stroke, whereas the activity remained stable. CONCLUSIONS: Lp-PLA2 mass is higher in ICH compared with ischemic stroke during the hyperacute stage. Lp-PLA2 activity is associated with stroke volume in ICH but not in ischemic stroke. This suggests that Lp-PLA2 mass and activity could provide different information in the hyperacute stage of stroke.

Gene-environment interactions in severe intraventricular hemorrhage of preterm neonates.

Intraventricular hemorrhage (IVH) of the preterm neonate is a complex developmental disorder, with contributions from both the environment and the genome. IVH, or hemorrhage into the germinal matrix of the developing brain with secondary periventricular infarction, occurs in that critical period of time before the 32nd to 33rd wk postconception and has been attributed to changes in cerebral blood flow to the immature germinal matrix microvasculature. Emerging data suggest that genes subserving coagulation, inflammatory, and vascular pathways and their interactions with environmental triggers may influence both the incidence and severity of cerebral injury and are the subject of this review. Polymorphisms in the Factor V Leiden gene are associated with the atypical timing of IVH, suggesting an as yet unknown environmental trigger. The methylenetetrahydrofolate reductase (MTHFR) variants render neonates more vulnerable to cerebral injury in the presence of perinatal hypoxia. The present study demonstrates that the MTHFR 677C>T polymorphism and low 5-min Apgar score additively increase the risk of IVH. Finally, review of published preclinical data suggests the stressors of delivery result in hemorrhage in the presence of mutations in collagen 4A1, a major structural protein of the developing cerebral vasculature. Maternal genetics and fetal environment may also play a role.

Hemorrhagic transformation during thrombolytic therapy and reperfusion: effects of age, blood pressure, and matrix metalloproteinases.

BACKGROUND: Despite the increasing use of thrombolytic therapy for acute ischemic stroke, hemorrhagic transformation remains a significant complication. Transformation appears to occur more frequently with age, diabetes, and hypertension, but clinical data are mixed. In addition to risk factors, matrix metalloproteinase expression mediates hemorrhage. We sought to test the effects of age, hypertension, and matrix metalloproteinases during recombinant tissue plasminogen activator (rt-PA) treatment in a standard model of filament occlusion of the middle cerebral artery. METHODS: We compared young and aged rats who were genetically predisposed to hypertension to similar and dissimilar strains to separate the effect of hypertension and age. RESULTS: Hemorrhagic transformation occurred significantly more frequently in chronically hypertensive animals-23 of 53 (44%) compared to 2 of 23 (9%) normotensive, genetically similar rats (Chi-square; P < .001; Mantel-Haenszel common odds ratio estimate 12.33 [95% confidence interval 2.7-57.0]). Hemorrhage rates were comparable in aged and young chronically hypertensive animals. Induced acute hypertension during reperfusion did not appear to alter rates of transformation. In hypertensive (n = 26) compared to genetically similar normotensive (n = 12) animals, rt-PA treatment increased mortality to 35% from 0% (Chi-square; P < .05), while hemorrhage occurred in 50% of the rt-PA-treated hypertensive subjects compared to 8% of the normotensive animals (Chi-square; P < .05). Two different inhibitors of matrix metalloproteinases significantly reduced mortality but not hemorrhage rates. CONCLUSIONS: Our data suggest for the first time an effect of chronic hypertension separate from age on the risk of hemorrhagic transformation. In addition, inhibitors of matrix metalloproteinases may protect the neurovascular unit directly, even without reducing hemorrhage risk. These findings will require additional research.

Microglial cell activation is a source of metalloproteinase generation during hemorrhagic transformation.

Hemorrhage and edema accompany evolving brain tissue injury after ischemic stroke. In patients, these events have been associated with metalloproteinase (MMP)-9 in plasma. Both the causes and cellular sources of MMP-9 generation in this setting have not been defined. MMP-2 and MMP-9 in nonhuman primate tissue in regions of plasma leakage, and primary murine microglia and astrocytes, were assayed by immunocytochemistry, zymography, and real-time RT-PCR. Ischemia-related hemorrhage was associated with microglial activation in vivo, and with the leakage of plasma fibronectin and vitronectin into the surrounding tissue. In strict serum-depleted primary cultures, by zymography, pro-MMP-9 was generated by primary murine microglia when exposed to vitronectin and fibronectin. Protease secretion was enhanced by experimental ischemia (oxygen-glucose deprivation, OGD). Primary astrocytes, on each matrix, generated only pro-MMP-2, which decreased during OGD. Microglia-astrocyte contact enhanced pro-MMP-9 generation in a cell density-dependent manner under normoxia and OGD. Compatible with observations in a high quality model of focal cerebral ischemia, microglia, but not astrocytes, respond to vitronectin and fibronectin, found when plasma extravasates into the injured region. Astrocytes alone do not generate pro-MMP-9. These events explain the appearance of MMP-9 antigen in association with ischemia-induced cerebral hemorrhage and edema.

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.

Evaluation of MTHFR C677T polymorphism in ischemic and hemorrhagic stroke patients. A case-control study in a Northern Indian population.

OBJECTIVE: The present study was aimed to evaluate MTHFR C677T gene polymorphism in patients with ischemic stroke (IS) and intracerebral hemorrhage (ICH) and compare it with controls. METHODS: 207 patient with IS and 215 with CT/MRI proven ICH were included and compared with 188 healthy controls. The stroke risk factors, location of IS, its vascular territory and in ICH the location of hematoma were noted. MTHFR C677T polymorphism was studied by polymerase chain reaction. RESULTS: Hypertension was present in 65.9% of ICH and 48.8% of IS. Other stroke risk factors were not significantly different. The frequency of the CC genotype in controls was 68.6%. CT in 28.7% and TT in 2.7%, whereas it was 75.3%, 20.5% and 4.2% in ICH and 66.2%, 39.4% and 2.4% respectively in IS. The frequency of these genotypes as well as allele frequency was not different in IS, ICH as compared to controls, however variant allele was more frequent in IS compared to ICH. Homocysteine level was higher in IS patients with variant genotype INTERPRETATION: MTHFR C677T gene polymorphism was neither associated with hemorrhagic nor ischemic stroke. However raised homocysteine levels were found to be associated with MTHFRC677-TT genotype in IS patients.

Matrix metalloproteinase 2 (MMP-2) degrades soluble vasculotropic amyloid-beta E22Q and L34V mutants, delaying their toxicity for human brain microvascular endothelial cells.

Patients carrying mutations within the amyloid-beta (Abeta) sequence develop severe early-onset cerebral amyloid angiopathy with some of the related variants manifesting primarily with hemorrhagic phenotypes. Matrix metalloproteases (MMPs) are typically associated with blood brain barrier disruption and hemorrhagic transformations after ischemic stroke. However, their contribution to cerebral amyloid angiopathy-related hemorrhage remains unclear. Human brain endothelial cells challenged with Abeta synthetic homologues containing mutations known to be associated in vivo with hemorrhagic manifestations (AbetaE22Q and AbetaL34V) showed enhanced production and activation of MMP-2, evaluated via Multiplex MMP antibody arrays, gel zymography, and Western blot, which in turn proteolytically cleaved in situ the Abeta peptides. Immunoprecipitation followed by mass spectrometry analysis highlighted the generation of specific C-terminal proteolytic fragments, in particular the accumulation of Abeta-(1-16), a result validated in vitro with recombinant MMP-2 and quantitatively evaluated using deuterium-labeled internal standards. Silencing MMP-2 gene expression resulted in reduced Abeta degradation and enhanced apoptosis. Secretion and activation of MMP-2 as well as susceptibility of the Abeta peptides to MMP-2 degradation were dependent on the peptide conformation, with fibrillar elements of AbetaE22Q exhibiting negligible effects. Our results indicate that MMP-2 release and activation differentially degrades Abeta species, delaying their toxicity for endothelial cells. However, taking into consideration MMP ability to degrade basement membrane components, these protective effects might also undesirably compromise blood brain barrier integrity and precipitate a hemorrhagic phenotype.

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.

Elevation of matrix metalloproteinases 3 and 9 in cerebrospinal fluid and blood in patients with severe traumatic brain injury.

OBJECTIVE: Traumatic brain injury (TBI) causes an increase in matrix metalloproteinases (MMPs), which are associated with neuroinflammation, blood-brain barrier disruption, hemorrhage, and cell death. We hypothesized that patients with TBI have an increase in MMPs in ventricular cerebrospinal fluid (CSF) and plasma. METHODS: Patients with TBI and a ventricular catheter were entered into the study. Samples of CSF and plasma were collected at the time of catheter placement and at 24 and 72 hours after admission. Seven TBI patients were entered into the study, with 6 having complete data for analysis. Only patients who had a known time of insult that fell within a 6-hour window from initial insult to ventriculostomy were accepted into the study. Control CSF came from ventricular fluid in patients undergoing shunt placement for normal pressure hydrocephalus. Both MMP-2 and MMP-9 were measured with gelatin zymography and MMP-3 with Western immunoblot. RESULTS: We found a significant elevation in the levels of the latent form of MMP-9 (92-kD) in the CSF obtained at the time of arrival (P < 0.05). Elevated levels of MMP-2 were detected in plasma at 72 hours, but not in the CSF. Using albumin from both CSF and blood, we calculated the MMP-9 index, which was significantly increased in the CSF, indicating endogenous MMP production. Western immunoblot showed elevated levels of MMP-3 in CSF at all times measured, whereas MMP-3 was not detected in the CSF of normal pressure hydrocephalus. CONCLUSION: We show that MMPs are increased in the CSF of TBI patients. Although the number of patients was small, the results were robust and clearly demonstrated increases in MMP-3 and MMP-9 in ventricular CSF in TBI patients compared with controls. Although these preliminary results will need to be replicated, we propose that MMPs may be important in blood-brain barrier opening and hemorrhage secondary to brain injury in patients.

Matrix metalloproteinase-3 (MMP3) and MMP9 genes and risk of myocardial infarction, ischemic stroke, and hemorrhagic stroke.

OBJECTIVE: We assessed the association of genetic variation in MMP3 and MMP9 with risk of myocardial infarction and stroke. METHODS: A case-control study was conducted among members of Group Health (GH), a large-integrated health care delivery system. Case subjects with incident non-fatal myocardial infarction (n=854), ischemic stroke (n=367), and hemorrhagic stroke (n=66) were identified and validated. A matched control group was selected from among GH members without myocardial infarction or stroke (n=2696). Haplotype-tagging sets of single-nucleotide polymorphisms (SNPs) in MMP3 and MMP9 were genotyped. RESULTS: MMP3 haplotype 2 was associated with reduced risk of myocardial infarction (adjusted odds ratio (OR) per copy=0.80, 95% confidence interval 0.66, 0.98) and increased risk of hemorrhagic stroke (OR=1.69, 95% confidence interval 1.05, 2.75). Results for MMP3 haplotype 2 and ischemic stroke resembled those for myocardial infarction but did not achieve statistical significance (OR=0.85, 95% confidence interval 0.64, 1.12). No individual SNP identified MMP3 haplotype 2, and none of the individual MMP3 SNPs were associated with myocardial infarction or stroke. MMP9 haplotypes or SNPs were not associated with myocardial infarction or stroke. CONCLUSIONS: MMP3 haplotype may predict both cardiac events and stroke.

Oxidative stress through activation of NAD(P)H oxidase in hypertensive mice with spontaneous intracranial hemorrhage.

We have developed an experimental model of spontaneous intracranial hemorrhage (ICH) in transgenic mice expressing human renin and human angiotensinogen (R+/A+) treated with high-salt diet and N(omega)-nitro-L-arginine methyl ester (L-NAME). We investigated whether oxidative stress is associated with spontaneous ICH in R+/A+ mice. R+/A+ mice on high-salt diet and L-NAME presented neurologic signs 57+/-13 (mean+/-s.e.m.) days after the start of treatment. Intracranial hemorrhage was shown with histologic examination. Levels of superoxide in brain homogenate were significantly increased in R+/A+ mice with ICH (118+/-10 RLU per sec per mg; RLU, relative light unit) compared with age-matched control mice (19+/-1) and R+/A+ mice without ICH (53+/-3). NAD(P)H oxidase activity was significantly higher in R+/A+ mice with ICH (34,933+/-2,420 RLU per sec per mg) than in control mice (4,984+/-248) and R+/A+ mice without ICH (15,069+/-917). These results suggest that increased levels of superoxide are due, at least in part, to increased NAD(P)H oxidase activity. Increased NAD(P)H oxidase activity preceded signs of ICH, and increased further when R+/A+ mice developed ICH. These findings suggest that oxidative stress may contribute to spontaneous ICH in chronic hypertension.

[Glutathione system in erythrocytes and blood plasma in strokes and dyscirculatory encephalopathy].

In dyscirculatory encephalopathy and moderate ischemic stroke there are single changes of components of glutathione metabolism. In moderate and severe ischemic stroke frequent and considerable changes have been revealed. Changes in hemorrhagic stroke are also expressed. An increase of activities of glutathione peroxidase and glutathione transferase is the most typical, rarely the increase of glutathione reductase and GSH is observed. The increase of enzymes activity was absent at the delayed oneset of treatment (more than 3 days) and in severe cases patients who died later. Glutathione system is important in the tolerance to cerebral ischemia.

Stromelysin-1 (MMP-3) is critical for intracranial bleeding after t-PA treatment of stroke in mice.

BACKGROUND: Tissue-type plasminogen activator (t-PA) is approved for treatment of ischemic stroke patients, but it may increase the risk of intracranial bleeding (ICB). Matrix metalloproteinases (MMPs), which can be activated through the plasminogen/plasmin system, may contribute to ICB after ischemic stroke. OBJECTIVES: To explore the contribution of plasminogen, MMP-3 and MMP-9 to ICB associated with t-PA treatment after ischemic stroke. METHODS: Using a thrombotic middle cerebral artery occlusion (MCA-O) model, ICB was studied in mice with genetic deficiencies of plasminogen (Plg(-/-)), stromelysin-1 (MMP-3(-/-)), or gelatinase B (MMP-9(-/-)) and their corresponding wild-type (WT) littermates. The induction of MMP-3 and MMP-9 was also studied in C57BL/6 WT mice. RESULTS: ICB induced by t-PA (10 mg kg(-1)) was significantly less than WT in Plg(-/-) (P < 0.05) and MMP-3(-/-) (P < 0.05) but not in MMP-9(-/-) mice. Furthermore, administration of the broad-spectrum MMP inhibitor GM6001 after t-PA treatment reduced ICB significantly (P < 0.05) in MMP-3(+/+) mice, but had no effect on MMP-3(-/-) mice. MMP-3 expression was significantly enhanced at the ischemic hemisphere; with placebo treatment, it was expressed only in neurons, whereas it was up-regulated in endothelial cells with t-PA treatment. Although MMP-9 expression was also significantly enhanced at the ischemic brain, the amount and the distribution were comparable in mice with and without t-PA treatment. CONCLUSIONS: Our data with gene-deficient mice thus suggest that plasminogen and MMP-3 are relatively more important than MMP-9 for the increased ICB induced by t-PA treatment of ischemic stroke.

Effect of neutrophil depletion on gelatinase expression, edema formation and hemorrhagic transformation after focal ischemic stroke.

BACKGROUND: While gelatinase (MMP-2 and -9) activity is increased after focal ischemia/reperfusion injury in the brain, the relative contribution of neutrophils to the MMP activity and to the development of hemorrhagic transformation remains unknown. RESULTS: Anti-PMN treatment caused successful depletion of neutrophils in treated animals. There was no difference in either infarct volume or hemorrhage between control and PMN depleted animals. While there were significant increases in gelatinase (MMP-2 and MMP-9) expression and activity and edema formation associated with ischemia, neutrophil depletion failed to cause any change. CONCLUSION: The main finding of this study is that, in the absence of circulating neutrophils, MMP-2 and MMP-9 expression and activity are still up-regulated following focal cerebral ischemia. Additionally, neutrophil depletion had no influence on indicators of ischemic brain damage including edema, hemorrhage, and infarct size. These findings indicate that, at least acutely, neutrophils are not a significant contributor of gelatinase activity associated with acute neurovascular damage after stroke.

Hematoma removal, heme, and heme oxygenase following hemorrhagic stroke.

The hemorrhagic strokes, intracerebral (ICH) and subarachnoid hemorrhage (SAH), often have poor outcomes. Indeed, the most common hemorrhagic stroke, ICH, has the highest mortality and morbidity rates of any stroke subtype. In this report, we discuss the evidence for the staging of red blood cell removal after ICH and the significance of control of this process. The protective effects of clinically relevant metalloporphyrin heme oxygenase inhibitors in experimental models of ICH and in superficial siderosis are also discussed. We also examine literature paradoxes related to both heme and heme oxygenase in various disorders of the central nervous system. Last, new data are presented that support the concept that heme, although primarily a pro-oxidant, can also have antioxidant properties.