Reduction of Tissue Plasminogen Activator-Induced Matrix Metalloproteinase-9 by Simvastatin in Astrocytes
Background and Purpose— Hemorrhagic conversion after tissue plasminogen activator (tPA) stroke therapy has been linked with elevations in matrix metalloproteinase-9 (MMP-9) at the neurovascular interface. Here, we test the idea that statins may directly ameliorate tPA-induced MMP-9 dysregulation.
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.
Thrombolysis with tissue plasminogen activator (tPA) can effectively restore blood flow in the ischemic brain but increases the risk of life-threatening intracerebral hemorrhage. Because the neurovascular protease matrix metalloproteinase-9 (MMP-9) mediates blood-brain barrier leakage and edema in stroke,1,2 an emerging hypothesis suggests that tPA upregulates MMP-9 after stroke, subsequently damaging the neurovascular matrix to cause hemorrhagic transformation.3–6 Therefore, strategies to reduce tPA-induced MMP-9 dysregulation may improve the safety and efficacy of tPA. Here, we tested the hypothesis that statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, can directly reduce tPA-induced MMP-9 levels in astrocytes.
Materials and Methods
Primary rat astrocyte cultures were prepared as described previously.7 Cells were treated with simvastatin (gift from James Liao and Michael Moskowitz) or Y-27632 (Calbiochem) for 4 hours and then incubated for 24 hours with recombinant human tPA (Activase; Genentech). Rho kinase signaling was assessed using Western blots to look for localization of Rho to the membrane fraction of cell lysates following standard techniques.
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.
Our rat cortical astrocytes primarily secreted the 88-kDa form of MMP-9. Exposure of rat cortical astrocytes to tPA (5 μg/mL) for 24 hours upregulated 88-kDa MMP-9 levels in conditioned media. Pretreatment with simvastatin (1 to 10 μmol/L) significantly ameliorated this response (Figure 1A and 1B). MMP-2 levels (both 72 and 66 kDa) were upregulated by tPA, but the response tended to be variable, and simvastatin treatment had no statistically significant effects (Figure 1C and 1D).
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).
Statins may be beneficial in ischemic stroke through their anti-inflammatory actions8 and can upregulate endothelial NO synthase and tPA in the cerebral endothelium, which improves cerebral perfusion9 and contributes to clot lysis,10 respectively. Our data point to another possible beneficial action of statins (ie, amelioration of tPA-induced MMP-9) that is known to damage the neurovascular unit and contribute to brain injury after stroke.1–6 Kilic et al recently showed that statins may reduce tPA-associated reperfusion injury in a mouse model of focal cerebral ischemia.11 More recently, Zhang et al showed that combination therapy with atorvastatin plus tPA decreased MMP-9 and extended the time window for thrombolysis in a rat embolic clot model.12 A caveat with in vivo models is that it may be difficult to distinguish direct statin effects on MMPs versus multifactorial statin effects on overall tissue injury. Our data show that simvastatin directly suppresses MMP-9 in cells. Although there are important differences between different types of statins, taken together, these data indicate that statins may be protective against tPA-induced MMP-9 deregulation and the associated hemorrhagic complications in stroke.
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.
Sources of Funding
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.
- Received March 2, 2006.
- Accepted March 30, 2006.
Asahi M, Wang X, Mori T, Sumii T, Jung JC, Moskowitz MA, Fini ME, Lo EH. Effects of matrix metalloproteinase-9 gene knock-out on the proteolysis of blood-brain barrier and white matter components after cerebral ischemia. J Neurosci. 2001; 21: 7724–7732.
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: 1759–1766.
Lo EH, Broderick JP, Moskowitz MA. tPA and proteolysis in the neurovascular unit. Stroke. 2004; 35: 354–356.
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: 598–603.
Sumii T, Lo EH. Involvement of matrix metalloproteinase in thrombolysis-associated hemorrhagic transformation after embolic focal ischemia in rats. Stroke. 2002; 33: 831–836.
Wang X, Mori T, Sumii T, Lo EH. Hemoglobin-induced cytotoxicity in rat cerebral cortical neurons: caspase activation and oxidative stress. Stroke. 2002; 33: 1882–1888.
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: 8880–8885.
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: 332–336.
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: 3486–3494.
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: 2251–2257.