Regulation of the endothelial plasminogen activator system by fluvastatin

Summary

Statins are cholesterol-lowering drugs that exert pleiotropic effects which include changes in the plasminogen activation (PA) system of endothelial cells (EC). It was the objective of this study to investigate the signal transduction pathways by which statins increase the expression of tissue-type PA (t-PA) and decrease PA inhibitor type 1 (PAI-1) in human umbilical vein EC. Fluvastatin treatment increased t-PA expression more than 10-fold and reduced PAI-1 expression up to fivefold. This effect was mimicked by geranylgeranyl transferase inhibition. The role of geranylgeranylated small G-proteins of the Rho family was assessed by adenovirus-mediated expression of dominant negative (DN) RhoA, Cdc42 and Rac1 and by siRNA-mediated suppression of these proteins. DN-Cdc42 and DN-Rac1, as well as siRNA for Cdc42, increased t-PA expression, while DN-RhoA and DN-Rac1 decreased PAI-1 expression. Latrunculin B, an inhibitor of actin polymerisation, in-creased t-PA mRNA and reduced PAI-1 mRNA to the same extent as fluvastatin. Inhibition of p38, as well as p38α or p38β siRNA, reversed the effects of fluvastatin on t-PA expression. Treatment with p38β siRNA partially reversed the effect of fluvastatin on PAI-1, whereas p38α siRNA had no significant effect. Inhibition of jun kinase reduced basal and fluvastatin-induced t-PA expression to the same extent and increased PAI-1. MEK/ERK inhibition had no effect. In human EC, the fluvastatin-induced increase in t-PA is mediated by Cdc42 and, as with t-PA induced by inhibition of actin polymerisation, requires activation of p38MAP kinase. The mechanisms by which fluvastatin treatment reduces PAI-1 are different from those that increase t-PA.

Keywords

Endothelial cells - statin - t-PA - PAI-1 - p38 - Rho family proteins - actin polymerization

^* These authors contributed equally to this article.

• References

• 1 Takemoto M, Liao JK. Pleiotropic effects of 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitors. Arterioscler Thromb Vasc Biol 2001; 21: 1712-1719.
• 2 Essig M, Nguyen G, Prié D. et al. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors increase fibrinolytic activity in rat aortic endothelial cells. Role of geranylgeranylation and Rho proteins. Circ Res 1998; 83: 683-690.
• 3 Mussoni L, Banfi C, Sironi L. et al. Fluvastatin inhibits basal and stimulated plasminogen activator inhibitor 1, but induces tissue type plasminogen activator in cultured human endothelial cells. Thromb Haemost 2000; 84: 59-64.
• 4 Bourcier T, Libby P. HMG CoA reductase inhibitors reduce plasminogen activator inhibitor-1 expression by human vascular smooth muscle and endothelial cells. Arterioscler Thromb Vasc Biol 2000; 20: 556-562.
• 5 Wiesbauer F, Kaun C, Zorn G. et al. HMG CoA reductase inhibitors affect the fibrinolytic system of human vascular cells in vitro: a comparative study using different statins. Br J Pharmacol 2002; 135: 284-292.
• 6 Vojtek AB, Cooper JA. Rho family members, activators of MAP kinase cascades. Cell 1995; 82: 527-529.
• 7 Dunoyer-Geindre S, Kruithof EK, Galve-de Rochemonteix B. et al. Localization of beta2-glycoprotein 1 in late endosomes of human endothelial cells. Thromb Haemost 2001; 85: 903-907.
• 8 Fish RJ, Kruithof EK. Short-term cytotoxic effects and long-term instability of RNAi delivered using lentiviral vectors. BMC Mol Biol 2004; 5: 9.
• 9 Huber D, Cramer EM, Kaufmann JE. et al. Tissue-type plasminogen activator (t-PA) is stored in Weibel-Palade bodies in human endothelial cells both in vitro and in vivo. Blood 2002; 99: 3637-3645.
• 10 Fish RJ, Yang H, Viglino C. et al. Fluvastatin inhibits regulated secretion of endothelial cell von Willebrand factor in response to diverse secretagogues. Biochem J 2007; 405: 597-604.
• 11 Siekmeier R, Lattke P, Mix C. et al. Dose dependency of fluvastatin pharmacokinetics in serum determined by reversed phase HPLC. J Cardiovasc Pharmacol Ther 2001; 6: 137-145.
• 12 Lobell RB, Omer CA, Abrams MT. et al. Evaluation of farnesyl:protein transferase and geranylgeranyl:protein transferase inhibitor combinations in preclinical models. Cancer Res. 2001; 61: 8758-8768.
• 13 Turner SJ, Zhuang S, Zhang T. et al. Effects of lovastatin on Rho isoform expression, activity, and association with guanine nucleotide dissociation inhibitors. Biochem Pharmacol 2008; 7: 405-413.
• 14 Heasman SJ, Ridley AJ. Mammalian Rho GTPases: new insights into their functions from in vivo studies. Nat Rev Mol Cell Biol 2008; 9: 690-701.
• 15 Bain J, Plater L, Elliott M. et al. The selectivity of protein kinase inhibitors: a further update. Biochem J 2007; 408: 297-315.
• 16 Ho TT, Merajver SD, Lapière CM. et al. RhoA-GDP regulates RhoB protein stability. Potential involvement of RhoGDIalpha. J Biol Chem. 2008; 283: 21588-21598.
• 17 Takeda K, Ichiki T, Tokunou T. et al. Critical role of Rho-kinase and MEK/ERK pathways for angiotensin II-induced plasminogen activator inhibitor type-1 gene expression. Arterioscler Thromb Vasc Biol 2001; 21: 868-873.
• 18 Kunieda Y, Nakagawa K, Nishimura H. et al. HMG CoA reductase inhibitor suppresses the expression of tissue factor and plasminogen activator inhibitor-1 induced by angiotensin II in cultured rat aortic endothelial cells. Thromb Res 2003; 110: 227-234.
• 19 Rikitake Y, Liao JK. Rho-kinase mediates hyperglycemia-induced plasminogen activator inhibitor-1 expression in vascular endothelial cells. Circulation 2005; 111: 3261-3268.
• 20 Kato T, Hashikabe H, Iwata C. et al. Statin blocks Rho/Rho-kinase signalling and disrupts the actin cytoskeleton: relationship to enhancement of LPS-mediated nitric oxide synthesis in vascular smooth muscle cells. Biochim Biophys Acta 2004; 1689: 267-272.
• 21 Yang C, Patel K, Harding P. et al. Regulation of TGF-beta1/MAPK-mediated PAI-1 gene expression by the actin cytoskeleton in human mesangial cells. Exp Cell Res. 2007; 313: 1240-1250.
• 22 Frigeri L, Apgar JR. The role of actin microfilaments in the down-regulation of the degranulation response in RBL-2H3 mast cells. J Immunol. 1999; 16: 2243-2250.
• 23 Providence KM, Kutz SM, Higgins PJ. Perturbation of the actin cytoskeleton induces PAI-1 gene expression in cultured epithelial cells independant of substrate anchorage. Cell Motil Cytoskeleton 1999; 42: 218-229.
• 24 Samarakoon R, Higgins PJ. MEK/ERK pathway mediates cell-shape-dependent plasminogen activator inhibitor type 1 gene expression upon drug-induced disruption of the microfilament and microtubule networks. J Cell Sci. 2002; 115: 3093-3103.
• 25 Kusterman G, Piette J, Legrand-Poels S. Actin-targeting natural compounds as tool to study the role of actin cytoskeleton in signal transduction. Biochem Pharmacol 2008; 76: 1310-1322.
• 26 Ulfhammer E, Larsson P, Karlsson L. et al. TNF-alpha mediated suppression of tissue type plasminogen activator expression in vascular endothelial cells is NF-kappaB- and p38 MAPK-dependent. J Thromb Haemost 2006; 4: 1781-1789.
• 27 Nakajima K, Suga H, Matsuno H. et al. Differential roles of MAP kinases in atorvastatin-induced VEGF release in cardiac myocytes. Life Sci 2006; 79: 1214-1220.
• 28 Pepper MS, Rosnoblet C, Di Sanza C. et al. Synergistic induction of t-PA by vascular endothelial growth factor and basic fibroblast growth factor and localization of t-PA to Weibel-Palade bodies in bovine microvascular endothelial cells. Thromb Haemost 2001; 86: 702-709.