Anne M. Romanic

Matrix Metalloproteinase Expression Increases After Cerebral Focal Ischemia in Rats: Inhibition of Matrix Metalloproteinase-9 Reduces Infarct Size

BACKGROUND AND PURPOSE Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade the extracellular matrix and are implicated in numerous pathological conditions including atherosclerosis, inflammation, and tumor growth and metastasis. In the brain, the endothelial cell wall, strengthened by tight junctions, defines the blood-brain barrier (BBB). The extracellular matrix molecules constitute the basement membrane underlying the vasculature and play a critical role in maintaining the integrity of the BBB. After focal stroke, there is a breakdown of the BBB with an associated increase in vascular permeability, inflammatory cell influx, and neuronal cell death. The present study was designed to investigate the effects of MMP expression after stroke. METHODS Focal stroke was produced by permanent middle cerebral artery occlusion (MCAO) in the rat, and MMP protein expression was measured by Western blot and zymogram analysis over a time course ranging from 6 hours to 30 days (n=32). Immunohistochemistry at 1 and 5 days (n=8 and 6, respectively) was also utilized to characterize the expression of several MMPs and related proteins after stroke, including their cellular source. To test the hypothesis that early increased MMP-9 expression is involved in ischemic brain injury, a neutralizing monoclonal antibody directed against MMP-9 was administered intravenously (n=7 per group) 1 hour before MCAO, and infarct size was measured 24 hours later. RESULTS MMP expression increased progressively over time after stroke. After 12 hours, significant (P<0.05) MMP-9 activity was observed that reached maximum levels by 24 hours (P<0.001), then persisted for 5 days at this level and returned to basal (zero) levels by 15 days. On the basis of morphological criteria, MMP-9 appeared to stain with endothelial cells and neutrophils identified both within and at the periphery of the infarct within 24 hours of focal ischemia. After 5 days, MMP-9 appeared to stain with macrophages present within the infarcted brain. MMP-2 activity was significantly (P<0.001) increased by 24 hours and was maximum after 5 days following MCAO. MMP-2 appeared to stain with macrophages present within the infarcted region. Unlike MMP-9 and MMP-2, tissue inhibitor of metalloproteinase-1 was identified at comparable levels in both control and ischemic tissue after MCAO. MMP-1 and MMP-3 could not be detected in the brain after focal stroke. When an MMP-9-neutralizing monoclonal antibody was administered systemically, animals exhibited significantly reduced infarct size (ie, a 30% reduction compared with non-immune antibody controls; P<0.05). CONCLUSIONS These results demonstrate that early increased MMP-9 expression in endothelial cells and infiltrating neutrophils is a significant response to cerebral focal ischemia and that selective inhibition of MMP-9 activity can significantly reduce brain injury after stroke.

Myocardial protection from ischemia/reperfusion injury by targeted deletion of matrix metalloproteinase-9

OBJECTIVE Matrix metalloproteinase-9 (MMP-9) activity is up regulated in the heart subjected to ischemic insult. Whether increased MMP-9 activity contributes to acute myocardial injury after ischemia-reperfusion remains unknown. To investigate the role of MMP-9 in myocardial infarction, we utilized a MMP-9 knockout mouse. METHODS AND RESULTS Standard homologous recombination in embryonic stem cells was used to generate a mouse lacking MMP-9. The left anterior descending coronary artery was occluded for 30 min followed by 24 h reperfusion, and the ischemic and infarct sizes were determined. Targeted deletion of MMP-9 protected the heart from no-flow ischemia-reperfusion-induced myocardial injury. The myocardial infarct size was reduced by 17.5% in MMP-9 heterozygotes (+/-) (P<0.01) and 35.4% in MMP-9 knockout (-/-) mice (P<0.01) versus the wild-type (+/+) mice, respectively. Analysis of MMP activity in myocardial extracts by zymography demonstrated that ischemia-reperfusion-induced expression of proMMP-9 and active MMP-9 was reduced by 77.8% (P<0.01) and 69.1% (P<0.001), respectively, in (+/-) mice compared to (+/+) mice, and was absent in (-/-) animals. The expression of TIMP-1, an endogenous inhibitor of MMP-9, was elevated 4.7-fold (P<0.05) and 21.4-fold (P<0.05) in the (+/-) and (-/-) mice, respectively, compared to (+/+) mice. Immunohistochemical analysis revealed that neutrophils were the primary cellular source of MMP-9, and less neutrophils were detected in the ischemic region of the heart following ischemia-reperfusion in (-/-) mice compared to (+/+) mice. Measurement of myeloperoxidase activity, a marker enzyme of neutrophils, demonstrated a 44% reduction in neutrophils infiltrated into the ischemic myocardium in the (-/-) mice compared to the (+/+) mice (P<0.05). CONCLUSION These results suggest that MMP-9 plays an important role in ischemia-reperfusion-induced myocardial infarction and MMP-9 could be a target for prevention or treatment of acute ischemic myocardial injury.

Activation of Peroxisome Proliferator–Activated Receptor-α Protects the Heart From Ischemia/Reperfusion Injury

Background—Peroxisome proliferator–activated receptor-&agr; (PPAR-&agr;) is expressed in the heart and regulates genes involved in myocardial fatty acid oxidation (FAO). The role of PPAR-&agr; in acute ischemia/reperfusion myocardial injury remains unclear. Methods and Results—The coronary arteries of male mice were ligated for 30 minutes. After reperfusion for 24 hours, ischemic and infarct sizes were determined. A highly selective and potent PPAR-&agr; agonist, GW7647, was administered by mouth for 2 days, and the third dose was given 1 hour before ischemia. GW7647 at 1 and 3 mg · kg−1 · d−1 reduced infarct size by 28% and 35%, respectively (P <0.01), and myocardial contractile dysfunction was also improved. Cardioprotection by GW7647 was completely abolished in PPAR-&agr;–null mice. Ischemia/reperfusion downregulated mRNA expression of cardiac PPAR-&agr; and FAO enzyme genes, decreased myocardial FAO enzyme activity and in vivo cardiac fat oxidation, and increased serum levels of free fatty acids. All of these changes were reversed by GW7647. Moreover, GW7647 attenuated ischemia/reperfusion-induced release of multiple proinflammatory cytokines and inhibited neutrophil accumulation and myocardial expression of matrix metalloproteinases-9 and -2. Furthermore, GW7647 inhibited nuclear factor-&kgr;B activation in the heart, accompanied by enhanced levels of inhibitor-&kgr;B&agr;. Conclusions—Activation of PPAR-&agr; protected the heart from reperfusion injury. This cardioprotection might be mediated through metabolic and antiinflammatory mechanisms. This novel effect of the PPAR-&agr; agonist could provide an added benefit to patients treated with PPAR-&agr; activators for dyslipidemia.

Activation of peroxisome proliferator-activated receptor-alpha protects the heart from ischemia/reperfusion injury.

Background—Peroxisome proliferator–activated receptor-&agr; (PPAR-&agr;) is expressed in the heart and regulates genes involved in myocardial fatty acid oxidation (FAO). The role of PPAR-&agr; in acute ischemia/reperfusion myocardial injury remains unclear. Methods and Results—The coronary arteries of male mice were ligated for 30 minutes. After reperfusion for 24 hours, ischemic and infarct sizes were determined. A highly selective and potent PPAR-&agr; agonist, GW7647, was administered by mouth for 2 days, and the third dose was given 1 hour before ischemia. GW7647 at 1 and 3 mg · kg−1 · d−1 reduced infarct size by 28% and 35%, respectively (P <0.01), and myocardial contractile dysfunction was also improved. Cardioprotection by GW7647 was completely abolished in PPAR-&agr;–null mice. Ischemia/reperfusion downregulated mRNA expression of cardiac PPAR-&agr; and FAO enzyme genes, decreased myocardial FAO enzyme activity and in vivo cardiac fat oxidation, and increased serum levels of free fatty acids. All of these changes were reversed by GW7647. Moreover, GW7647 attenuated ischemia/reperfusion-induced release of multiple proinflammatory cytokines and inhibited neutrophil accumulation and myocardial expression of matrix metalloproteinases-9 and -2. Furthermore, GW7647 inhibited nuclear factor-&kgr;B activation in the heart, accompanied by enhanced levels of inhibitor-&kgr;B&agr;. Conclusions—Activation of PPAR-&agr; protected the heart from reperfusion injury. This cardioprotection might be mediated through metabolic and antiinflammatory mechanisms. This novel effect of the PPAR-&agr; agonist could provide an added benefit to patients treated with PPAR-&agr; activators for dyslipidemia.

Matrix Metalloproteinase Expression Increases After Cerebral Focal Ischemia in Rats

BACKGROUND AND PURPOSE Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade the extracellular matrix and are implicated in numerous pathological conditions including atherosclerosis, inflammation, and tumor growth and metastasis. In the brain, the endothelial cell wall, strengthened by tight junctions, defines the blood-brain barrier (BBB). The extracellular matrix molecules constitute the basement membrane underlying the vasculature and play a critical role in maintaining the integrity of the BBB. After focal stroke, there is a breakdown of the BBB with an associated increase in vascular permeability, inflammatory cell influx, and neuronal cell death. The present study was designed to investigate the effects of MMP expression after stroke. METHODS Focal stroke was produced by permanent middle cerebral artery occlusion (MCAO) in the rat, and MMP protein expression was measured by Western blot and zymogram analysis over a time course ranging from 6 hours to 30 days (n=32). Immunohistochemistry at 1 and 5 days (n=8 and 6, respectively) was also utilized to characterize the expression of several MMPs and related proteins after stroke, including their cellular source. To test the hypothesis that early increased MMP-9 expression is involved in ischemic brain injury, a neutralizing monoclonal antibody directed against MMP-9 was administered intravenously (n=7 per group) 1 hour before MCAO, and infarct size was measured 24 hours later. RESULTS MMP expression increased progressively over time after stroke. After 12 hours, significant (P<0.05) MMP-9 activity was observed that reached maximum levels by 24 hours (P<0.001), then persisted for 5 days at this level and returned to basal (zero) levels by 15 days. On the basis of morphological criteria, MMP-9 appeared to stain with endothelial cells and neutrophils identified both within and at the periphery of the infarct within 24 hours of focal ischemia. After 5 days, MMP-9 appeared to stain with macrophages present within the infarcted brain. MMP-2 activity was significantly (P<0.001) increased by 24 hours and was maximum after 5 days following MCAO. MMP-2 appeared to stain with macrophages present within the infarcted region. Unlike MMP-9 and MMP-2, tissue inhibitor of metalloproteinase-1 was identified at comparable levels in both control and ischemic tissue after MCAO. MMP-1 and MMP-3 could not be detected in the brain after focal stroke. When an MMP-9-neutralizing monoclonal antibody was administered systemically, animals exhibited significantly reduced infarct size (ie, a 30% reduction compared with non-immune antibody controls; P<0.05). CONCLUSIONS These results demonstrate that early increased MMP-9 expression in endothelial cells and infiltrating neutrophils is a significant response to cerebral focal ischemia and that selective inhibition of MMP-9 activity can significantly reduce brain injury after stroke.

Matrix Metalloproteinase Expression Increases After Cerebral Focal Ischemia in Rats : Inhibition of Matrix Metalloproteinase-9 Reduces Infarct Size Editorial Comment: Inhibition of Matrix Metalloproteinase-9 Reduces Infarct Size

BACKGROUND AND PURPOSE Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade the extracellular matrix and are implicated in numerous pathological conditions including atherosclerosis, inflammation, and tumor growth and metastasis. In the brain, the endothelial cell wall, strengthened by tight junctions, defines the blood-brain barrier (BBB). The extracellular matrix molecules constitute the basement membrane underlying the vasculature and play a critical role in maintaining the integrity of the BBB. After focal stroke, there is a breakdown of the BBB with an associated increase in vascular permeability, inflammatory cell influx, and neuronal cell death. The present study was designed to investigate the effects of MMP expression after stroke. METHODS Focal stroke was produced by permanent middle cerebral artery occlusion (MCAO) in the rat, and MMP protein expression was measured by Western blot and zymogram analysis over a time course ranging from 6 hours to 30 days (n=32). Immunohistochemistry at 1 and 5 days (n=8 and 6, respectively) was also utilized to characterize the expression of several MMPs and related proteins after stroke, including their cellular source. To test the hypothesis that early increased MMP-9 expression is involved in ischemic brain injury, a neutralizing monoclonal antibody directed against MMP-9 was administered intravenously (n=7 per group) 1 hour before MCAO, and infarct size was measured 24 hours later. RESULTS MMP expression increased progressively over time after stroke. After 12 hours, significant (P<0.05) MMP-9 activity was observed that reached maximum levels by 24 hours (P<0.001), then persisted for 5 days at this level and returned to basal (zero) levels by 15 days. On the basis of morphological criteria, MMP-9 appeared to stain with endothelial cells and neutrophils identified both within and at the periphery of the infarct within 24 hours of focal ischemia. After 5 days, MMP-9 appeared to stain with macrophages present within the infarcted brain. MMP-2 activity was significantly (P<0.001) increased by 24 hours and was maximum after 5 days following MCAO. MMP-2 appeared to stain with macrophages present within the infarcted region. Unlike MMP-9 and MMP-2, tissue inhibitor of metalloproteinase-1 was identified at comparable levels in both control and ischemic tissue after MCAO. MMP-1 and MMP-3 could not be detected in the brain after focal stroke. When an MMP-9-neutralizing monoclonal antibody was administered systemically, animals exhibited significantly reduced infarct size (ie, a 30% reduction compared with non-immune antibody controls; P<0.05). CONCLUSIONS These results demonstrate that early increased MMP-9 expression in endothelial cells and infiltrating neutrophils is a significant response to cerebral focal ischemia and that selective inhibition of MMP-9 activity can significantly reduce brain injury after stroke.

Escherichia coli lipopolysaccharide potentiation and inhibition of rat neonatal microglia superoxide anion generation : Correlation with prior lactic dehydrogenase, nitric oxide, tumor necrosis factor-α, thromboxane B2, and metalloprotease release

The effects of lipopolysaccharide (LPS) on the central nervous system, one of the first organs to be affected by sepsis, are still incompletely understood. Rat microglia (BMphi) constitute the main leukocyte-dependent source of reactive oxygen species in the central nervous system. The in vitro effect of LPS on agonist-stimulated superoxide (O2-) generation from BMphi appears controversial. Our purpose was to determine the time- and concentration-dependent effect of Escherichia coil LPS on phorbol-12 myristate 13-acetate-stimulated O2- generation from BMphi. Our results demonstrate that BMphi O2- generation in vitro peaked 17 h after stimulation of with .3 ng/mL LPS. Furthermore, stimulation of BMphi with LPS for 17 h resulted in the following concentration-dependent responses: .1-1 ng/mL LPS induced no prior mediator generation but potently enhanced subsequent phorbol-12 myristate 13-acetate-stimulated O2- generation; 3-10 ng/mL LPS caused nitric oxide, tumor necrosis factor-alpha (TNF-alpha), thromboxane B2 and matrix metalloproteinase-9 release although partially inhibiting ensuing phorbol-12 myristate 13-acetate-stimulated O2- generation; 30-100 ng/mL LPS, maximized nitric oxide, TNF-alpha, thromboxane B2, matrix metalloproteinase-9 generation with concomitant lactic dehydrogenase release although strongly deactivating successive phorbol-12 myristate 13-acetate-stimulated O2 production. Our in vitro studies suggest that enhanced release of these four mediators (nitric oxide, TNF-alpha, thromboxane B2, and matrix metalloproteinase-9) during stimulation of BMphi with LPS might play a critical role in the subsequent ability of BMphi to generate O2- in vivo. Potential clinical implications of our findings are suggested by the fact that LPS levels similar to the ones used in this study have been observed in cerebrospinal fluid both in Gram-negative meningitis and sepsis.

Effect of a short-term in vitro exposure to the marine toxin domoic acid on viability, tumor necrosis factor-alpha, matrix metalloproteinase-9 and superoxide anion release by rat neonatal microglia

BackgroundThe excitatory amino acid domoic acid, a glutamate and kainic acid analog, is the causative agent of amnesic shellfish poisoning in humans. No studies to our knowledge have investigated the potential contribution to short-term neurotoxicity of the brain microglia, a cell type that constitutes circa 10% of the total glial population in the brain. We tested the hypothesis that a short-term in vitro exposure to domoic acid, might lead to the activation of rat neonatal microglia and the concomitant release of the putative neurotoxic mediators tumor necrosis factor-α (TNF-α), matrix metalloproteinases-2 and-9 (MMP-2 and -9) and superoxide anion (O2-).ResultsIn vitro, domoic acid [10 μM-1 mM] was significantly neurotoxic to primary cerebellar granule neurons. Although neonatal rat microglia expressed ionotropic glutamate GluR4 receptors, exposure during 6 hours to domoic acid [10 μM-1 mM] had no significant effect on viability. By four hours, LPS (10 ng/mL) stimulated an increase in TNF-α mRNA and a 2,233 % increase in TNF-α protein In contrast, domoic acid (1 mM) induced a slight rise in TNF-α expression and a 53 % increase (p < 0.01) of immunoreactive TNF-α protein. Furthermore, though less potent than LPS, a 4-hour treatment with domoic acid (1 mM) yielded a 757% (p < 0.01) increase in MMP-9 release, but had no effect on MMP-2. Finally, while PMA (phorbol 12-myristate 13-acetate) stimulated O2- generation was elevated in 6 hour LPS-primed microglia, a similar pretreatment with domoic acid (1 mM) did not prime O2- release.ConclusionsTo our knowledge this is the first experimental evidence that domoic acid, at in vitro concentrations that are toxic to neuronal cells, can trigger a release of statistically significant amounts of TNF-α and MMP-9 by brain microglia. These observations are of considerable pathophysiological significance because domoic acid activates rat microglia several days after in vivo administration.

New Strategies in Drug Discovery

Gene identification followed by determination of the expression of genes in a given disease and understanding of the function of the gene products is central to the drug discovery process. The ability to associate a specific gene with a disease can be attributed primarily to the extraordinary progress that has been made in the areas of gene sequencing and information technologies. Selection and validation of novel molecular targets have become of great importance in light of the abundance of new potential therapeutic drug targets that have emerged from human gene sequencing. In response to this revolution within the pharmaceutical industry, the development of high-throughput methods in both biology and chemistry has been necessitated. Further, the successful translation of basic scientific discoveries into clinical experimental medicine and novel therapeutics is an increasing challenge. As such, a new paradigm for drug discovery has emerged. This process involves the integration of clinical, genetic, genomic, and molecular phenotype data partnered with cheminformatics. Central to this process, the data generated are managed, collated, and interpreted with the use of informatics. This review addresses the use of new technologies that have arisen to deal with this new paradigm.