Role of matrix metalloproteinases in delayed cortical responses after stroke

Matrix metalloproteinases (MMPs) are zinc-endopeptidases with multifactorial actions in central nervous system (CNS) physiology and pathology. Accumulating data suggest that MMPs have a deleterious role in stroke. By degrading neurovascular matrix, MMPs promote injury of the blood-brain barrier, edema and hemorrhage. By disrupting cell-matrix signaling and homeostasis, MMPs trigger brain cell death. Hence, there is a movement toward the development of MMP inhibitors for acute stroke therapy. But MMPs may have a different role during delayed phases after stroke. Because MMPs modulate brain matrix, they may mediate beneficial plasticity and remodeling during stroke recovery. Here, we show that MMPs participate in delayed cortical responses after focal cerebral ischemia in rats. MMP-9 is upregulated in peri-infarct cortex at 7-14 days after stroke and is colocalized with markers of neurovascular remodeling. Treatment with MMP inhibitors at 7 days after stroke suppresses neurovascular remodeling, increases ischemic brain injury and impairs functional recovery at 14 days. MMP processing of bioavailable VEGF may be involved because inhibition of MMPs reduces endogenous VEGF signals, whereas additional treatment with exogenous VEGF prevents MMP inhibitor-induced worsening of infarction. These data suggest that, contrary to MMP inhibitor therapies for acute stroke, strategies that modulate MMPs may be needed for promoting stroke recovery.     

Peroxynitrite decomposition catalyst prevents matrix metalloproteinase activation and neurovascular injury after prolonged cerebral ischemia in rats

Matrix metalloproteinases (MMPs) play an important role in reperfusion-induced brain injury following ischemia. To define the effects of peroxynitrite decomposition catalyst on MMP activation and neurovascular reperfusion injury, 5,10,15,20-tetrakis (2,4,6-trimethyl-3,5-disulfonatophenyl)-porphyrin iron (III) (FeTMPyP) was administered intravenously 30?min prior to reperfusion following a middle cerebral artery occlusion. Activation of MMP was assessed by in situ and gel zymography. Neurovascular injury was assessed using endothelial barrier antigen, collagen IV immunohistochemistry and Cresyl violet staining. Results were compared with sham and ischemia alone groups. We found that administration of FeTMPyP just before reperfusion after ischemia inhibited MMP-9 activation and total MMP-2 increases in the cortex and decreased active MMP-9 along with the total amounts of active MMP-9 and active MMP-2 in the striatum. Reperfusion-induced injury to the basal lamina of collagen IV-immunopositive microvasculature and neural cells in cortex and striatum was ameliorated by FeTMPyP. Losses of blood vessel endothelium produced by ischemia or reperfusion were also decreased in the cortex. These results suggest that administration of FeTMPy prior to reperfusion decreases MMP activation and neurovascular injury after prolonged cerebral ischemia. This strategy may be useful for future therapies targeted at preventing breakdown of the blood-brain barrier and hemorrhagic transformation.     

Lipoprotein receptor-mediated induction of matrix metalloproteinase by tissue plasminogen activator

Although thrombolysis with tissue plasminogen activator (tPA) is a stroke therapy approved by the US Food and Drug Administration, its efficacy may be limited by neurotoxic side effects. Recently, proteolytic damage involving matrix metalloproteinases (MMPs) have been implicated. In experimental embolic stroke models, MMP inhibitors decreased cerebral hemorrhage and injury after treatment with tPA. MMPs comprise a family of zinc endopeptidases that can modify several components of the extracellular matrix. In particular, the gelatinases MMP-2 and MMP-9 can degrade neurovascular matrix integrity. MMP-9 promotes neuronal death by disrupting cell-matrix interactions, and MMP-9 knockout mice have reduced blood-brain barrier leakage and infarction after cerebral ischemia. Hence it is possible that tPA upregulates MMPs in the brain, and that subsequent matrix degradation causes brain injury. Here we show that tPA upregulates MMP-9 in cell culture and in vivo. MMP-9 levels were lower in tPA knockouts compared with wild-type mice after focal cerebral ischemia. In human cerebral microvascular endothelial cells, MMP-9 was upregulated when recombinant tPA was added. RNA interference (RNAi) suggested that this response was mediated by the low-density lipoprotein receptor-related protein (LRP), which avidly binds tPA and possesses signaling properties. Targeting the tPA-LRP signaling pathway in brain may offer new approaches for decreasing neurotoxicity and improving stroke therapy.     

Green tea polyphenol (−)-epigallocatechin gallate reduces matrix metalloproteinase-9 activity following transient focal cerebral ischemia

Green tea polyphenol (?)-epigallocatechin gallate (EGCG) has been reported to reduce neuronal damage after cerebral ischemic insult. EGCG is known to reduce matrix metalloproteinase (MMP) activity. MMP can play an important role in the pathophysiology of neurological disorders including cerebral ischemia. The purpose of the current study was to investigate whether EGCG shows an inhibitory effect on MMP activity and neural tissue damage following transient focal cerebral ischemia. In the present study, C57BL/6 mice were subjected to 80 min of focal ischemia induced by middle cerebral artery occlusion (MCAO). Animals were killed 24 h after ischemia. EGCG (50 mg/kg) was administered intraperitoneally immediately after ischemia. Gelatin gel zymography showed an increase in the active form of MMP-9 after ischemia. EGCG reduced ischemia-induced up-regulation of the active form of MMP-9. In in situ zymography, EGCG reduced up-regulation of gelatinase activity induced by cerebral ischemia. Co-incubation with EGCG reduced gelatinase activity directly in postischemic brain section. In 2,3,5-triphenyltetrazolium chloride (TTC) assay, brain infarction was remarkable in the middle cerebral artery territory after focal cerebral ischemia. In EGCG-treated mice, infarct volume was significantly reduced compared with vehicle-treated mice. These results demonstrate that EGCG, a green tea polyphenol, may reduce up-regulation of MMP-9 activity and neuronal damage following transient focal cerebral ischemia. In addition to its antioxidant effect, MMP-9 inhibition might be a possible mechanism potentially involved in the neuroprotective effect of a green tea polyphenol, EGCG.     

Acute connexin43 temporal and spatial expression in response to ischemic stroke

Connexin43 (Cx43) gap junctions expressed in astrocytes can significantly impact neuronal survival in stroke. However, little is known regarding Cx43 spatial and temporal expression during the initial stages of brain ischemia. Using immunohistochemistry and Western blot analysis, we examined Cx43 spatial and temporal expression as a function of neuronal injury within the first 24 h after permanent middle cerebral artery occlusion (pMCAO). Western blot analysis showed a significant increase in Cx43 protein expression in the core ischemic area at 2 and 3 h after pMCAO. However, after 6 h of pMCAO Cx43 levels were significantly reduced. This reduction was due to cell death and concomitant Cx43 degradation in the expanding focal ischemic region, while the peri-infarct zone revealed intense Cx43 staining. The neuronal cell-death marker Fluoro-Jade C labeled injured neurons faintly at 1 h post-pMCAO with a time-dependent increase in both intensity and size of punctate staining. In addition, decreased microtubule-associated protein 2 (MAP2) immunoreactivity and thionin staining similarly indicated cell damage beginning at 1 h after pMCAO. Taken together, Cx43 expression is sensitive to neuronal injury and can be detected as early as 2 h post-pMCAO. These findings underscore Cx43 gap junction as a potential early target for therapeutic intervention in ischemic stroke.     

Normobaric hyperoxia inhibits NADPH oxidase-mediated matrix metalloproteinase-9 induction in cerebral microvessels in experimental stroke

Matrix metalloproteinase-9 (MMP-9) and NADPH oxidase contribute to blood-brain barrier (BBB) disruption after ischemic stroke. We have previously shown that normobaric hyperoxia (NBO) treatment reduces MMP-9 and oxygen free radical generation in ischemic brain. In this study, we tested the hypothesis that NBO protects the BBB through inhibiting NADPH oxidase-mediated MMP-9 induction in transient focal cerebral ischemia. Male Sprague-Dawley rats (n = 69) were given NBO (95% O2) or normoxia (21% O2) during 90-min filament occlusion of the middle cerebral artery. Cerebral microvessels were isolated for analyzing MMP-9 and NADPH oxidase. BBB damage was non-invasively quantified with magnetic resonance imaging. In normoxic rats, both NADPH oxidase catalytic subunit gp91(phox) and MMP-9 expression were up-regulated in ischemic hemispheric microvessels after 90-min middle cerebral artery occlusion with 22.5 h reperfusion. Inhibition of NADPH oxidase with apocynin reduced the MMP-9 increase, indicating a causal link between NADPH oxidase-derived superoxide and MMP-9 induction. NBO treatment inhibited gp91(phox) expression, NADPH oxidase activity, and MMP-9 induction, which led to significantly less BBB damage and brain edema in the ischemic brain. These results suggest that gp91(phox) containing NADPH oxidase plays an important role in MMP-9 induction in ischemic BBB microvasculature, and that NBO treatment may attenuate MMP-9 induction and brain edema through inhibiting NADPH oxidase after transient cerebral ischemia.     

Effect of Baicalin on Matrix Metalloproteinase-9 Expression and Blood–Brain Barrier Permeability Following Focal Cerebral Ischemia in Rats

Focal cerebral ischemia results in an increased expression of matrix metalloproteinase-9 (MMP-9), which induces vasogenic brain edema via disrupting the blood–brain barrier (BBB) integrity. Recent studies from our laboratory showed that baicalin reduces ischemic brain damage by inhibiting inflammatory reaction and neuronal apoptosis in a rat model of focal cerebral ischemia. In the present study, we first explored the effect of baicalin on the neuronal damage, brain edema and BBB permeability, then further investigated its potential mechanisms. Sprague–Dawley rats underwent permanent middle cerebral artery occlusion (MCAO). Baicalin was administrated by intraperitoneally injected twice at 2 and 12 h after the onset of MCAO. Neuronal damage, brain edema and BBB permeability were measured 24 h following MCAO. Expression of MMP-9 protein and mRNA were determined by western blot and RT–PCR, respectively. Expression of tight junction protein (TJP) occludin was detected by western blot. Neuronal damage, brain edema and BBB permeability were significantly reduced by baicalin administration following focal cerebral ischemia. Elevated expression of MMP-9 protein and mRNA were significantly down-regulated by baicalin administration. In addition, MCAO caused the decreased expression of occludin, which was significantly up-regulated by baicalin administration. Our study suggested that baicalin reduces MCAO-induced neuronal damage, brain edema and BBB permeability, which might be associated with the inhibition of MMP-9 expression and MMP-9-mediated occludin degradation.     

EMMPRIN (basigin/CD147) is involved in the morphogenesis of tooth germ in mouse molars

The pattern of gene expression for extracellular matrix metalloproteinase inducer (EMMPRIN) was revealed in the tooth germ of mouse mandibular molars using quantitative real-time PCR. In situ hybridization and immunohistochemical study demonstrated the characteristic distribution of EMMPRIN in the different stages of tooth germ development. To investigate the functional role played by EMMPRIN in tooth germ development, EMMPRIN siRNA interference approach was carried out in cultured mouse mandibles at embryonic day 11.0 (E11.0). The results showed that EMMPRIN siRNA-treated explants exhibited a marked growth inhibition of tooth germ compared to the control and scrambled siRNA-treated explants. Meanwhile, a significant increase in MT1-MMP mRNA expression and a reduction in MMP-2, MMP-3, MMP-9, MMP-13 and MT2-MMP mRNA expression were observed in the mouse mandibles following EMMPRIN abrogation. The current results indicate that EMMPRIN could thus be involved in the early stage of tooth germ development and morphogenesis, possibly by regulating the expression of MMP genes.     

N-Myc Downstream-Regulated Gene 2 (Ndrg2) Is Involved in Ischemia–Hypoxia-Induced Astrocyte Apoptosis: a Novel Target for Stroke Therapy

Nearly all clinical trials that have attempted to develop effective strategies against ischemic stroke have failed, excluding those for thrombolysis, and most of these trials focused only on preventing neuronal loss. However, astrocytes have gradually become a target for neuroprotection in stroke. In previous studies, we showed that the newly identified molecular N-myc downstream-regulated gene 2 (Ndrg2) is specifically expressed in astrocytes in the brain and involved in some neurodegenerative diseases. However, the role of NDRG2 in ischemic stroke remained unclear. In this study, we investigated the role of NDRG2 in middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia and in oxygen–glucose deprivation (OGD)-induced cellular apoptosis in the M1800 astrocyte cell line. NDRG2 mRNA and protein expression began to increase at 6 and 2 h after reperfusion and peaked at 24 h in the ischemic penumbra and in M1800 cells, as detected by RT-PCR and Western blotting. Double immunofluorescence staining showed that the number of apoptotic cells increased as the NDRG2-positive signal increased and that the NDRG2 signal was sometimes co-localized with TUNEL-positive cells and translocated from the cytoplasm to the nucleus in both the ischemic penumbra and the M1800 cells. Using a lentivirus, we successfully constructed two stable astrocytic cell lines in which NDRG2 expression was significantly up- or down-regulated. NDRG2 silencing had a proliferative effect and reduced the percentage of apoptotic cells, reactive oxygen species (ROS) production, and cleaved Caspase-3 protein expression following OGD, whereas NDRG2 over-expression had the opposite effects. In conclusion, NDRG2 is involved in astrocyte apoptosis following ischemic–hypoxic injury, and inhibiting NDRG2 expression significantly reduces ROS production and astrocyte apoptosis. These findings provide insight into the role of NDRG2 in ischemic–hypoxic injury and provide potential targets for future clinical therapies for stroke.     

A new penumbra: transitioning from injury into repair after stroke

The penumbra is an area of brain tissue that is damaged but not yet dead after focal ischemia. The existence of a penumbra implies that therapeutic salvage is theoretically possible after stroke. The first decade of penumbral science investigated the ischemic regulation of electrophysiology, cerebral blood flow and metabolism. The second decade advanced our understanding of molecular mechanisms that mediate penumbral cell death. And the third decade saw the rapid development of clinical neuroimaging tools that are now increasingly applied in stroke patients. But how can we look ahead as we move into the fourth decade of penumbra research? This author speculates that a paradigm shift is needed. Most molecular targets for therapy have biphasic roles in stroke pathophysiology. During the acute phase, these targets mediate injury. During the recovery phase, the same mediators contribute to neurovascular remodeling. It is this boundary zone that comprises the new penumbra, and future investigations should dissect where, when and how damaged brain makes the transition from injury into repair.     

MMP-9 Inhibition: a Therapeutic Strategy in Ischemic Stroke

Ischemic stroke is a leading cause of disability worldwide. In cerebral ischemia there is an enhanced expression of matrix metallo-proteinase-9 (MMP-9), which has been associated with various complications including excitotoxicity, neuronal damage, apoptosis, blood–brain barrier (BBB) opening leading to cerebral edema, and hemorrhagic transformation. Moreover, the tissue plasminogen activator (tPA), which is the only US-FDA approved treatment of ischemic stroke, has a brief 3 to 4 h time window and it has been proposed that detrimental effects of tPA beyond the 3 h since the onset of stroke are derived from its ability to activate MMP-9 that in turn contributes to the breakdown of BBB. Therefore, the available literature suggests that MMP-9 inhibition can be of therapeutic importance in ischemic stroke. Hence, combination therapies of MMP-9 inhibitor along with tPA can be beneficial in ischemic stroke. In this review we will discuss the current status of various strategies which have shown neuroprotection and extension of thrombolytic window by directly or indirectly inhibiting MMP-9 activity. In the introductory part of the review, we briefly provide an overview on ischemic stroke, commonly used models of ischemic stroke and a role of MMP-9 in ischemia. In next part, the literature is organized as various approaches which have proven neuroprotective effects through direct or indirect decrease in MMP-9 activity, namely, using biotherapeutics, involving MMP-9 gene inhibition using viral vectors; using endogenous inhibitor of MMP-9, repurposing of old drugs such as minocycline, new chemical entities like DP-b99, and finally other approaches like therapeutic hypothermia.     

Inhibition of MMP-9 by a selective gelatinase inhibitor protects neurovasculature from embolic focal cerebral ischemia

ABSTRACT: BACKGROUND: Cerebral ischemia has been shown to induce activation of matrix metalloproteinases (MMPs), particularly MMP-9, which is associated with impairment of the neurovasculature, resulting in blood-brain barrier breakdown, hemorrhage and neurodegeneration. We previously reported that the thiirane inhibitor SB-3CT, which is selective for gelatinases (MMP-2 and 9), could antagonize neuronal apoptosis after transient focal cerebral ischemia. RESULTS: Here, we used a fibrin-rich clot to occlude the middle cerebral artery (MCA) and assessed the effects of SB-3CT on the neurovasculature. Results show that neurobehavioral deficits and infarct volumes induced by embolic ischemia are comparable to those induced by the filament-occluded transient MCA model. Confocal microscopy indicated embolus-blocked brain microvasculature and neuronal cell death. Post-ischemic SB-3CT treatment attenuated infarct volume, ameliorated neurobehavioral outcomes, and antagonized the increases in levels of proform and activated MMP-9. Embolic ischemia caused degradation of the neurovascular matrix component laminin and tight-junction protein ZO-1, contraction of pericytes, and loss of lectin-positive brain microvessels. Despite the presence of the embolus, SB-3CT mitigated these outcomes and reduced hemorrhagic volumes. Interestingly, SB-3CT treatment for seven days protected against neuronal laminin degradation and protected neurons from ischemic cell death. CONCLUSION: These results demonstrate considerable promise for the thiirane class of selective gelatinase inhibitors as potential therapeutic agents in stroke therapy.     

CD38 exacerbates focal cytokine production, postischemic inflammation and brain injury after focal cerebral ischemia

Background

Converging evidence suggests that inflammatory processes significantly influence brain injury and clinical impairment in ischemic stroke. Although early studies suggested a key role of lymphocytes, recent data has emphasized the orchestrating function of innate immunity, i.e., macrophages and microglia. The bifunctional receptor and ectoenzyme CD38 synthesizes calcium-mobilizing second messengers (e.g., cyclic ADP-ribose), which have been shown to be necessary for activation and migration of myeloid immune cells. Therefore, we investigated the dynamics of CD38 in stroke and the impact of CD38-deficiency on cytokine production, inflammation and cerebral damage in a mouse model of cerebral ischemia-reperfusion.

Methodology/Principal Findings

We show that the local expression of the chemokine MCP-1 was attenuated in CD38-deficient mice compared with wildtype mice after focal cerebral ischemia and reperfusion. In contrast, no significant induction of MCP-1 expression was observed in peripheral blood after 6 hours. Flow cytometry analysis revealed less infiltrating macrophages and lymphocytes in the ischemic hemisphere of CD38-deficient mice, whereas the amount of resident microglia was unaltered. An up-regulation of CD38 expression was observed in macrophages and CD8⁺ cells after focal cerebral ischemia in wildtype mice, whereas CD38 expression was unchanged in microglia. Finally, we demonstrate that CD38-deficiency decreases the cerebral ischemic injury and the persistent neurological deficit after three days of reperfusion in this murine temporary middle cerebral artery occlusion (tMCAO) model.

Conclusion/Significance

CD38 is differentially regulated following stroke and its deficiency attenuates the postischemic chemokine production, the immune cell infiltration and the cerebral injury after temporary ischemia and reperfusion. Therefore CD38 might prove a therapeutic target in ischemic stroke.     

Absence of glutathione peroxidase-1 exacerbates cerebral ischemia-reperfusion injury by reducing post-ischemic microvascular perfusion

Mice deficient in the anti-oxidant enzyme glutathione peroxidase-1 (Gpx1) have a greater susceptibility to cerebral injury following a localized ischemic event. Much of the response to ischemia-reperfusion is caused by aberrant responses within the microvasculature, including inflammation, diminished endothelial barrier function (increased vascular permeability), endothelial activation, and reduced microvascular perfusion. However, the role of Gpx1 in regulating these responses has not been investigated. Wild-type and Gpx1-/- mice underwent focal cerebral ischemia via mid-cerebral artery occlusion followed by measurement of cerebral perfusion via laser Doppler and intravital microscopy. Post-ischemic brains in wild-type mice displayed significant deficit in microvascular perfusion. However, in Gpx1-/- mice, the deficit in cerebral blood flow was significantly greater than that in wild-type mice, and this was associated with significant increase in infarct size and increased vascular permeability. Ischemia-reperfusion also resulted in expression of matrix metalloproteinase-9 (MMP-9) in endothelial cells. The absence of Gpx1 was associated with marked increase in pro-MMP-9 expression as well as potentiated MMP-9 activity. Pre-treatment of Gpx1-/- mice with the anti-oxidant ebselen restored microvascular perfusion, limited the induction and activation of MMP-9, and attenuated the increases in infarct size and vascular permeability. These findings demonstrate that the anti-oxidant function of Gpx1 plays a critical role in protecting the cerebral microvasculature against ischemia-reperfusion injury by preserving microvascular perfusion and inhibiting MMP-9 expression.     

Prolonged Expression of Interferon-Inducible Protein-10 in Ischemic Cortex After Permanent Occlusion of the Middle Cerebral Artery in Rat

Abstract: Focal cerebral ischemia elicits local inflammatory reaction as demonstrated by the accumulation of inflammatory cells and mediators in the ischemic brain. Interferon-inducible protein-10 (IP-10) is a member of the C-X-C chemokine family that possesses potent chemoattractant actions for monocytes, T cells, and smooth muscle cells. To investigate a potential role of IP-10 in focal stroke, we studied the temporal expression of IP-10 mRNA after occlusion of the middle cerebral artery in rat by means of northern analysis. IP-10 mRNA expression after focal stroke demonstrated a unique biphasic profile, with a marked increase early at 3 h (4.9-fold over control; p < 0.01), a peak level at 6 h (14.5-fold; p < 0.001) after occlusion of the middle cerebral artery, and a second wave induction 10–15 days after ischemic injury (7.2- and 9.3-fold increase for 10 and 15 days, respectively; p < 0.001). In situ hybridization confirmed the induced expression of IP-10 mRNA and revealed its spatial distribution after focal stroke. Immunohistochemical studies demonstrated the expression of IP-10 peptide in neurons (3–12 h) and astroglial cells (6 h to 15 days) of the ischemic zone. To explore further the potential role of IP-10 in focal stroke, we demonstrated a dose-dependent chemotactic action of IP-10 on C6 glial cells and enhanced attachment of rat cerebellar granule neurons. Taken together, the data suggest that ischemia induces IP-10, which may play a pleiotropic role in prolonged leukocyte recruitment, astrocyte migration/activation, and neuron attachment/sprouting after focal stroke.     

Cerebral ischaemia and matrix metalloproteinase‐9 modulate the angiogenic function of early and late outgrowth endothelial progenitor cells

The enhancement of endogenous angiogenesis after stroke will be critical in neurorepair therapies where endothelial progenitor cells (EPCs) might be key players. Our aim was to determine the influence of cerebral ischaemia and the role of matrix metalloproteinase-9 (MMP-9) on the angiogenic function of EPCs. Permanent focal cerebral ischaemia was induced by middle cerebral artery (MCA) occlusion in MMP-9/knockout (MMP-9/KO) and wild-type (WT) mice. EPCs were obtained for cell counting after ischaemia (6 and 24 hrs) and in control animals. Matrigel™ assays and time-lapse imaging were conducted to monitor angiogenic function of WT and MMP9-deficient EPCs or after treatment with MMP-9 inhibitors. Focal cerebral ischaemia increased the number of early EPCs, while MMP-9 deficiency decreased their number in non-ischaemic mice and delayed their release after ischaemia. Late outgrowth endothelial cells (OECs) from ischaemic mice shaped more vessel structures than controls, while MMP-9 deficiency reduced the angiogenic abilities of OECs to form vascular networks, in vitro. Treatment with the MMP inhibitor GM6001 and the specific MMP-9 inhibitor I also decreased the number of vessel structures shaped by both human and mouse WT OECs, while exogenous MMP-9 could not revert the impaired angiogenic function in MMP-9/KO OECs. Finally, time-lapse imaging showed that the extension of vascular networks was influenced by cerebral ischaemia and MMP-9 deficiency early during the vascular network formation followed by a dynamic vessel remodelling. We conclude that focal cerebral ischaemia triggers the angiogenic responses of EPCs, while MMP-9 plays a key role in the formation of vascular networks by EPCs.