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(Stroke. 2006;37:1910.)
© 2006 American Heart Association, Inc.
Research Reports |
From the Neuroprotection Research Laboratory (S.W., S.R.L., S.Z.G., W.J.K., X.W., E.H.L.), Departments of Radiology and Neurology, Massachusetts General Hospital, and Program in Neuroscience, Harvard Medical School, Boston, Mass; Department of Life Science (S.R.L.), Cheju National University, Korea; Mount Sinai School of Medicine (S.W.), New York, NY; and Vall deHebron Hospital (J.M.), Barcelona, Spain.
Correspondence to Eng H. Lo, Neuroprotection Research Laboratory, MGH East 149-2401, Charlestown, MA 02129. E-mail Lo{at}helix.mgh harvard.edu
| Abstract |
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Methods Recombinant human tPA (5 µg/mL) was added to primary rat cortical astrocytes. Zymography was used to quantify MMP-9 levels in conditioned media. Effects of simvastatin or the Rho kinase inhibitor Y-27632 were assessed by pretreating cells before tPA exposure.
Results Simvastatin (1 to 10 µmol/L) significantly reduced tPA-induced MMP-9 in cortical astrocytes. This effect may be mediated via the Rho kinase pathway because tPA-induced activation of Rho signaling was suppressed by simvastatin, and tPA-induced MMP-9 levels were similarly reduced by the Rho kinase inhibitor Y-27632 (1 to 10 µmol/L).
Conclusions Statins reduce tPA-induced MMP-9 dysregulation by inhibiting the Rho signaling pathway. Statins may ameliorate tPA-associated MMP imbalances in stroke.
Key Words: hemorrhage stroke
| Introduction |
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| Materials and Methods |
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Gelatin Zymography
Gelatin zymography was performed as described previously.4 Human MMP-2 and MMP-9 standards (Chemicon) were used as positive controls. Gelatin zymography was quantified (n
4 per group) as the optical density (mean±SEM) and expressed as a ratio to the loaded positive controls. Data were analyzed using ANOVA and Tukey honestly significant difference tests. P<0.05 was considered significant.
| Results |
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Although statins can modulate multiple intracellular pathways, recent data suggest that inhibition of Rho kinase signaling may be an important downstream effect. Astrocytes that were exposed to tPA showed an activation in the Rho kinase pathway (Figure 2). Simvastatin prevented tPA-induced Rho activation (Figure 2). Consistent with this biochemical result, pretreatment with the potent and specific Rho kinase inhibitor Y-27632 (1 to 10 µmol/L) significantly ameliorated the tPA-induced MMP-9 response (Figure 3A and 3B). tPA-induced 72-kDa MMP-2 levels were also significantly decreased (Figure 3C), but 66-kDa MMP-2 levels were statistically unaffected by Rho kinase inhibition (Figure 3D).
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| Discussion |
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Cardiovascular benefits of statins were historically ascribed to their cholesterol-lowering actions. However, it is now recognized that statins modulate many other cell signaling pathways, including the Rho/ROCK pathway.13 In our model system, tPA activated Rho signaling, simvastatin prevented this effect, and the Rho kinase inhibitor Y-27632 downregulated MMP-9. Thus, the amelioration of tPA-induced MMP-9 by simvastatin in our study likely occurs via the Rho pathway.
Further studies are warranted to investigate the effects of statins on all cells of the neurovascular unit14 and determine whether specific statins or newly developed Rho kinase inhibitors15 can indeed suppress tPA-induced MMP imbalances and improve combination therapies for stroke.
| Acknowledgments |
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This work was supported by National Institutes of Health grants R01-NS37074, R01-NS48422, and P50-NS10828, a scientist development grant to X.W., a Howard Hughes Medical Research training fellowship to S.W., and a grant from the Korea Research Foundation to W.J.K.
Disclosures
None.
Received March 2, 2006; accepted March 30, 2006.
| References |
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2. Montaner J, Alvarez-Sabin J, Molina C, Angles A, Abilleira S, Arenillas J, Gonzalez MA, Monasterio J. Matrix metalloproteinase expression after human cardioembolic stroke: temporal profile and relation to neurological impairment. Stroke. 2001; 32: 17591766.
3. Lo EH, Broderick JP, Moskowitz MA. tPA and proteolysis in the neurovascular unit. Stroke. 2004; 35: 354356.
4. Wang X, Lee SR, Arai K, Tsuji K, Rebeck GW, Lo EH. Lipoprotein receptor-mediated induction of matrix metalloproteinase by tissue plasminogen activator. Nat Med. 2003; 9: 13131317.[CrossRef][Medline] [Order article via Infotrieve]
5. Montaner J, Molina CA, Monasterio J, Abilleira S, Arenillas JF, Ribo M, Quintana M, Alvarez-Sabin J. Matrix metalloproteinase-9 pretreatment level predicts intracranial hemorrhagic complications after thrombolysis in human stroke. Circulation. 2003; 107: 598603.
6. Sumii T, Lo EH. Involvement of matrix metalloproteinase in thrombolysis-associated hemorrhagic transformation after embolic focal ischemia in rats. Stroke. 2002; 33: 831836.
7. Wang X, Mori T, Sumii T, Lo EH. Hemoglobin-induced cytotoxicity in rat cerebral cortical neurons: caspase activation and oxidative stress. Stroke. 2002; 33: 18821888.
8. Menge T, Hartung HP, Stuve O. Statins-a cure-all for the brain? Nat Rev Neurosci. 2005; 6: 325331.[CrossRef][Medline] [Order article via Infotrieve]
9. Endres M, Laufs U, Huang Z, Nakamura T, Huang P, Moskowitz MA, Liao JK. Stroke protection by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide synthase. Proc Natl Acad Sci U S A. 1998; 95: 88808885.
10. Asahi M, Huang Z, Thomas S, Yoshimura S, Sumii T, Mori T, Qiu J, Amin-Hanjani S, Huang PL, Liao JK, Lo EH, Moskowitz MA. Protective effects of statins involving both eNOS and tPA in focal cerebral ischemia. J Cereb Blood Flow Metab. 2005; 25: 722729.[CrossRef][Medline] [Order article via Infotrieve]
11. Kilic E, Kilic U, Matter CM, Luscher TF, Bassetti CL, Hermann DM. Aggravation of focal cerebral ischemia by tissue plasminogen activator is reversed by 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitor but does not depend on endothelial no synthase. Stroke. 2005; 36: 332336.
12. Zhang L, Zhang ZG, Ding GL, Jiang Q, Liu X, Meng H, Hozeska A, Zhang C, Li L, Morris D, Zhang RL, Lu M, Chopp M. Multitargeted effects of statin-enhanced thrombolytic therapy for stroke with recombinant human tissue-type plasminogen activator in the rat. Circulation. 2005; 112: 34863494.
13. Liao JK. Isoprenoids as mediators of the biological effects of statins. J Clin Invest. 2002; 110: 285288.[CrossRef][Medline] [Order article via Infotrieve]
14. Lo EH, Dalkara T, Moskowitz MA. Mechanisms, challenges and opportunties in stroke. Nat Rev Neurosci. 2003; 4: 399415.[Medline] [Order article via Infotrieve]
15. Rikitake Y, Kim HH, Huang Z, Seto M, Yano K, Asano T, Moskowitz MA, Liao JK. Inhibition of Rho kinase (ROCK) leads to increased cerebral blood flow and stroke protection. Stroke. 2005; 36: 22512257.
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